Compositions and methods for treating obesity and related disorders by characterizing and restoring mammalian bacterial microbiota

ABSTRACT

The present invention relates to characterizing changes in mammalian intestinal microbiota associated with associated with high-fat and low-fat diets and with diets containing hydroxypropylmethylcellulose (HPMC) and related methods for diagnosing, preventing and treating obesity and related conditions such as metabolic syndrome and diabetes mellitus. Therapeutic methods of the invention involve the use of probiotics, and/or prebiotics, and/or narrow spectrum antibiotics/anti-bacterial agents that are capable of restoring healthy mammalian bacterial intestinal microbiota.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Divisional application of U.S. patent applicationSer. No. 13/214,034, filed on Aug. 19, 2011, now U.S. Pat. No.9,386,793, which claims the benefit of U.S. Provisional PatentApplication No. 61/375,678, filed on Aug. 20, 2010, both of whichapplications are incorporated herein by reference in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

Research and development leading to certain aspects of the presentinvention were supported, in part, by grants 1UL1RR029893 andR01DK098989 from the National Institutes of Health. Accordingly, theU.S. government may have certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to characterizing changes in mammalianintestinal microbiota associated with high-fat and low-fat diets andwith diets containing hydroxypropylmethylcellulose (HPMC) and relatedmethods for diagnosing, preventing and treating obesity and relatedconditions such as metabolic syndrome and diabetes mellitus. Therapeuticmethods of the invention involve the use of probiotics, and/orprebiotics, and/or narrow spectrum antibiotics/anti-bacterial agentsthat are capable of restoring healthy mammalian bacterial intestinalmicrobiota.

BACKGROUND OF THE INVENTION

Obesity has become widespread with increases in prevalence across alldeveloped nations (Bouchard, C (2000) N Engl J Med. 343, 1888-9).According to the Center for Disease Control (CDC), over 60% of theUnited States population is overweight, and greater than 30% are obese.For affected persons, the problem often begins in childhood, andcontinues for life. Major contributors are believed to be increasedconsumption of high calorie foods and a more sedentary life style.However, neither of these alone or together are sufficient to explainthe rise in obesity and subsequent or concomitant obesity-relateddisorders, such as, e.g., type II diabetes mellitus, metabolic syndrome,hypertension, cardiac pathology, and non-alcoholic fatty liver disease.According to the National Institute of Diabetes, Digestive and KidneyDiseases (NIDDK) approximately 280,000 deaths annually are directlyrelated to obesity. The NIDDK further estimated that the direct cost ofhealthcare in the U.S. associated with obesity is $51 billion. Inaddition, Americans spend $33 billion per year on weight loss products.The prevalence of obesity continues to rise at alarming rates.

It is estimated that between 20-25% of American adults (about 47million) have metabolic syndrome, a complex condition associated with anincreased risk of vascular disease. Metabolic syndrome is also known asSyndrome X, metabolic syndrome X, insulin resistance syndrome, orReaven's syndrome. Metabolic syndrome is generally believed to be acombination of disorders that affect a large number of people in aclustered fashion. The symptoms and features of the syndrome include atleast three of the following conditions: diabetes mellitus type II;impaired glucose tolerance or insulin resistance; high blood pressure;central obesity and difficulty losing weight; high cholesterol; combinedhyperlipidemia; including elevated LDL; decreased HDL; elevatedtriglycerides; and fatty liver (especially in concurrent obesity).Insulin resistance is typical of metabolic syndrome and leads to severalof its features, including glucose intolerance, dyslipidemia, andhypertension. Obesity is commonly associated with the syndrome as isincreased abdominal girth, highlighting the fact that abnormal lipidmetabolism likely contributes to the underlying pathophysiology ofmetabolic syndrome.

Metabolic syndrome was codified in the United States with thepublication of the National Cholesterol Education Program AdultTreatment Panel III (ATP III) guidelines in 2001. On a physiologicbasis, insulin resistance appears to be responsible for the syndrome.However, insulin resistance can be defined in a myriad of differentways, including impaired glucose metabolism (reduced clearance ofglucose and/or the failure to suppress glucose production), theinability to suppress lipolysis in tissues, defective protein synthesis,altered cell differentiation, aberrant nitric oxide synthesis affectingregional blood flow, as well as abnormal cell cycle control andproliferation, all of which have been implicated in the cardiovasculardisease associated with metabolic syndrome. At least at present, thereis no obvious molecular mechanism causing the syndrome, probably becausethe condition represents a failure of one or more of the manycompensatory mechanisms that are activated in response to energy excessand the accumulation of fat.

Individuals at risk for metabolic syndrome include those who exhibitcentral obesity with increased abdominal girth (due to excess visceraladiposity) of about more than 35 inches in women and more than 40 inchesin men. Individuals at risk for metabolic syndrome also include thosethat have a BMI greater than or equal to 30 kg/M2 and may also haveabnormal levels of nonfasting glucose, lipids, and blood pressure.

Although certain bacterial associations have been examined for these andrelated conditions, the role of bacterial microbiota in these conditionshas not been clearly understood or appreciated. Thus, there remains aneed for methods for diagnosing, treating and preventing conditions suchas obesity, metabolic syndrome, insulin-deficiency or insulin-resistancerelated disorders, glucose intolerance, diabetes mellitus, non-alcoholicfatty liver, abnormal lipid metabolism, atherosclerosis, and relateddisorders.

The average human body, consisting of about 10¹³ cells, has about tentimes that number of microorganisms. The ˜10¹⁴ microbes that live in andon each of our bodies belong to all three domains of life onearth—bacteria, archaea and eukarya. The major sites for our indigenousmicrobiota are the intestinal tract, skin and mucosal surfaces such asnasal mucosa and vagina as well as the oropharynx. By far, the largestbacterial populations are in the colon. Bacteria make up most of theflora in the colon and 60% of the dry mass of feces. Probably more than1000 different species live in the gut. However, it is probablethat >90% of the bacteria come from less than 50 species. Fungi andprotozoa also make up a part of the gut flora, but little is known abouttheir activities. While the microbiota is highly extensive, it is barelycharacterized. Consequently, the Roadmap of the National Institutes ofHealth (NIH) includes the “Human Microbiome Project” to bettercharacterize our microbial communities and the genes that they harbor(our microbiome) and better understand its relation to both human healthand disease. Reviewed in Dethlefsen et al., Nature, 2007, 449:811-818;Turnbaugh et al., Nature, 2007, 449:804-810; Ley et al., Cell, 2006,124:837-848.

Studies show that the relationship between gut flora and humans is notmerely commensal (a non-harmful coexistence), but rather often is amutualistic, symbiotic relationship. Although animals can survive withno gut flora, the microorganisms perform a host of useful functions,such as training the immune system, preventing growth of harmfulspecies, regulating the development of the gut, fermenting unused energysubstrates, metabolism of glycans and amino acids, synthesis of vitamins(such as biotin and vitamin K) and isoprenoids, biotransformation ofxenobiotics, and producing hormones to direct the host to store fats.See, e.g., Gill et al., Science. 2006, 312:1355-1359; Zaneveld et al.,Curr. Opin. Chem. Biol., 2008, 12(1):109-114; Guarner, Digestion, 2006,73:5-12; Li et al., Proc. Natl. Acad. Sci. USA, 2008, 105:2117-2122;Hooper, Trends Microbiol., 2004, 12:129-134; Mazmanian et al., Cell,2005, 122:107-118; Rakoff-Nahoum et al., Cell, 2004, 118:229-241. It istherefore believed that changes in the composition of the gut microbiotacould have important health effects (Dethlefsen et al., PLoS Biology,2008, 6(11):2383-2400). Indeed, a correlation between obesity andchanges in gut microbiota has been observed (Ley et al., Proc Natl AcadSci USA, 2005; 102:11070-11075; Bäackhed et al., Proc Natl Acad Sci USA,2004; 101:15718-15723). Furthermore, in certain conditions, somemicrobial species are thought to be capable of directly causing diseaseby causing infection or increasing cancer risk for the host (O'Keefe etal., J Nutr. 2007; 137:175S-182S; McGarr et al., J Clin Gastroenterol.,2005; 39:98-109).

Substantial number of species in vertebrate microbiota is very hard toculture and analyze via traditional cultivation-based studies (Turnbaughet al., Nature, 2007, 449:804-810; Eckburg et al., Science, 2005,308:1635-1638). In contrast, broad-range PCR primers targeted to highlyconserved regions makes possible the amplification of small subunit rRNAgene (16S rDNA) sequences from all bacterial species (Zoetendal et al.,(2006) Mol Microbiol 59, 1639-1650), and the extensive and rapidlygrowing 16S rDNA database facilitates identification of sequences to thespecies or genus level (Schloss and Handelsman, (2004) Microbiol MolBiol Rev 68, 686-691). Such techniques can also be used for identifyingbacterial species in complex environmental niches (Smit et al., (2001)Appl Environ Microbiol 67, 2284-2291), including the human mouth,esophagus, stomach, intestine, feces, skin, and vagina, and for clinicaldiagnosis (Harris and Hartley, (2003) J Med Microbiol 52, 685-691;Saglani et al., (2005) Arch Dis Child 90, 70-73).

Much of the microbiota is conserved from human to human, at least at thelevel of phylum and genus (for a general description of human microbiotasee, e.g., Turnbaugh et al., Nature 2007; 449:804-810; Ley et al.,Nature 2006; 444:1022-1023; Gao et al., Proc Natl Acad Sci USA 2007;104:2927-32; Pei et al., Proc Natl Acad Sci USA 2004; 101:4250-4255;Eckburg et al., Science 2005; 308:1635-1638; Bik et al., Proc Natl AcadSci USA 2006; 103:732-737). A major source of the human microbiota isfrom one's mother (for a summary of typical maternal colonizationpatterns see, e.g., Palmer et al., Plos Biology 2007; 5:e177; Raymond etal., Emerg Infect Dis 2004; 10:1816-21), and to a lesser extent fromone's father and siblings (for examples of typical colonization patternssee, e.g., Raymond et al., Emerg Infect Dis 2004; 10:1816-21; Raymond etal., Plos One 2008; 3:e2259; Goodman et al., Am J Epidemiol 1996;144:290-299; Goodman et al., Lancet 2000; 355:358-362). However, many ofthe natural mechanisms for the transmission of these indigenousorganisms across generations and between family members have diminishedwith socioeconomic development. The impediments include: childbirth bycaesarian section, reduced breast-feeding, smaller family size (fewersiblings), reduced household crowding with shared beds, utensils,in-door plumbing.

It has been known for more than 50 years that the administration of lowdoses of antibiotics promotes the growth of farm animals. See, e.g.,Jukes, Bioscience 1972; 22: 526-534; Jukes (1955) Antibiotics inNutrition. New York, N.Y., USA: Medical Encyclopedia; Feighner andDashkevicz, Appl. Environ. Microbiol., 1987, 53: 331-336; McEwen andFedorka-Cray, Clin. Infect. Dis., 2002, 34 (Suppl 3): S93-S106).

The mechanism for this widespread phenomenon has not been establishedbut because of the activity of anti-bacterial but not anti-fungalagents, it can be ascertained to be anti-bacterial.

The vertebrate intestinal tract has a rich component of cells involvedin immune responses. The nature of the microbiota colonizingexperimental animals or humans affects the immune responses of thepopulations of reactive host cells (see, e.g., Ando et al., Infectionand Immunity 1998; 66:4742-4747; Goll et al., Helicobacter. 2007;12:185-92; Lundgren et al., Infect Immun. 2005; 73:523-531).

The vertebrate intestinal tract also is a locus in which hormones areproduced. In mammals, many of these hormones related to energyhomeostasis (including insulin, glucagon, leptin, and ghrelin) areproduced by organs of the intestinal tract (see, e.g., Mix et al., Gut2000; 47:481-6; Kojima et al., Nature 1999; 402:656-60; Shak et al.,Obesity Surgery 2008; 18(9):1089-96; Roper et al., Journal of ClinicalEndocrinology & Metabolism 2008; 93:2350-7; Francois et al., Gut 2008;57:16-24; Cummings and Overduin, J Clin Invest 2007; 117:13-23; Bado etal., Nature 1998; 394:790-793).

Changing of the microbiota of the intestinal tract appears to affect thelevels of some of these hormones (see, e.g., Breidert et al., Scand JGastroenterol 1999; 34:954-61; Liew et al., Obes. Surg. 2006; 16:612-9;Nwokolo et al., Gut. 2003; 52, 637-640; Kinkhabwala et al.,Gastroenterology 132:A208). The hormones affect immune responses (see,e.g., Matarese et al., J Immunol 2005; 174:3137-3142; Matsuda et al., J.Allergy Clin. Immunol. 2007; 119, S174) and adiposity (see, e.g., Tschopet al., Nature 2000; 407:908-13).

Hydroxypropylmethylcellulose (HPMC) is modified cellulose fiber thatproduces viscous solutions in the gastrointestinal tract. It has beendemonstrated that high viscosity (HV) HPMC consumed as part of a mealreduced peak blood glucose concentrations in subjects with type 2diabetes compared with a cellulose control (Reppas et al., Diabetes Res.Clin. Pract., 1993, 22:61-9). It has been further demonstrated that HPMCreduced weight gain and insulin resistance in diet-induced obese miceand syrian hamsters fed a high fat (HF) diet similar in fat content tothe American diet. (Hung et al., J Diab 2009; 1(3):194-206); Kim et al.,FASEB J., 2009, Meeting Abstracts, Abstract 212.2).

PCT Pat. Appl. Publ. Nos. WO 2008/051793 and WO 2008/051794 disclose theuse of HPMC and other water-soluble and water-insoluble cellulosederivatives for preventing or treating metabolic syndrome and relatedconditions. See also U.S. Pat. Nos. 5,576,306; 5,585,366; 6,899,892;5,721,221. PCT Pat. Appl. Publ. No. WO 2004/022074 discloses the use ofa composition comprising a non-glucose carbohydrate and soluble fiber ora mixture of pectin and soluble fiber for controlling metabolicsyndrome, diabetes mellitus and obesity, and for the promotion of weightloss or maintenance of the desired body weight.

SUMMARY OF THE INVENTION

As specified in the Background section above, there is a great need inthe art to understand the impact that mammalian bacterial microbiota hason development of obesity and related disorders such as metabolicsyndrome, diabetes mellitus, insulin-deficiency or insulin-resistancerelated disorders, glucose intolerance, non-alcoholic fatty liver,abnormal lipid metabolism, and atherosclerosis. There is further a greatneed in the art to employ such knowledge in development of newtherapeutics to treat these and related disorders.

The present invention addresses these and other needs by characterizingspecific diet-induced-obesity-associated changes in mammalian bacterialmicrobiota and by providing related diagnostic and therapeutic methodsand probiotic and prebiotic compositions. The present invention furtherprovides novel prebiotic compositions based on a surprising finding thatcellulose ethers with a beta 1,4 linkage of anhydrous glucose units havea prebiotic effect although they are known to be substantiallynon-fermentable and non-digestible materials in the digestive tract ofmammals.

In one aspect, the invention provides a method for diagnosingpredisposition to a disease in a mammal selected from the groupconsisting of obesity, metabolic syndrome, diabetes mellitus,insulin-deficiency related disorders, insulin-resistance relateddisorders, glucose intolerance, non-alcoholic fatty liver, abnormallipid metabolism, and atherosclerosis, said method comprising

-   (a) measuring the populations of Firmicutes and/or Bacteroidetes in    the ileal microbiota of the mammal;-   (b) measuring the populations of Firmicutes and/or Bacteroidetes in    the ileal microbiota of a healthy control;-   (c) comparing the populations measured in steps (a) and (b), and-   (d) determining that the mammal has a predisposition to the disease    if the populations of Firmicutes and/or Bacteroidetes in the ileal    microbiota of the mammal are increased as compared to the healthy    control.

In a related aspect, the invention provides a method for promotingweight loss in a mammal comprising administering to the mammal atherapeutically effective amount of a probiotic composition, whereinsaid probiotic composition lowers the populations of Firmicutes and/orBacteroidetes in the ileal microbiota of the mammal.

In another related embodiment, the invention provides a method forpreventing or treating a disease in a mammal selected from the groupconsisting of obesity, metabolic syndrome, diabetes mellitus,insulin-deficiency related disorders, insulin-resistance relateddisorders, glucose intolerance, non-alcoholic fatty liver, abnormallipid metabolism, and atherosclerosis, said method comprisingadministering to the mammal a therapeutically effective amount of aprobiotic composition, wherein said probiotic composition lowers thepopulations of Firmicutes and/or Bacteroidetes in the ileal microbiotaof the mammal.

In another embodiment, the invention provides a method for promotingweight loss in a mammal comprising administering to the mammal atherapeutically effective amount of a prebiotic composition, whereinsaid prebiotic composition lowers the populations of Firmicutes and/orBacteroidetes in the ileal microbiota of the mammal.

In a further embodiment, the invention provides a method for preventingor treating a disease in a mammal selected from the group consisting ofobesity, metabolic syndrome, diabetes mellitus, insulin-deficiencyrelated disorders, insulin-resistance related disorders, glucoseintolerance, non-alcoholic fatty liver, abnormal lipid metabolism, andatherosclerosis, said method comprising administering to the mammal atherapeutically effective amount of a prebiotic composition, whereinsaid prebiotic composition lowers the populations of Firmicutes and/orBacteroidetes in the ileal microbiota of the mammal.

In a separate embodiment, the invention provides a method of loweringpopulations of Firmicutes and/or Bacteroidetes in the ileal microbiotaof a mammal comprising administering to the mammal a prebioticcomposition.

In another aspect, the invention provides a method for diagnosingpredisposition to a disease in a mammal selected from the groupconsisting of obesity, metabolic syndrome, diabetes mellitus,insulin-deficiency related disorders, insulin-resistance relateddisorders, glucose intolerance, non-alcoholic fatty liver, abnormallipid metabolism, and atherosclerosis, said method comprising

-   (a) measuring the populations of Firmicutes in the cecal and/or    fecal microbiota of the mammal;-   (b) measuring the populations of Firmicutes in the cecal and/or    fecal microbiota of a healthy control;-   (c) comparing the populations measured in steps (a) and (b), and-   (d) determining that the mammal has a predisposition to the disease    if the populations of Firmicutes in the cecal and/or fecal    microbiota of the mammal are increased as compared to the healthy    control.

In a related aspect, the invention provides a method for diagnosingpredisposition to a disease in a mammal selected from the groupconsisting of obesity, metabolic syndrome, diabetes mellitus,insulin-deficiency related disorders, insulin-resistance relateddisorders, glucose intolerance, non-alcoholic fatty liver, abnormallipid metabolism, and atherosclerosis, said method comprising

-   (a) measuring the ratio of the populations of Firmicutes to the    populations of Eubacteria (F/E ratio=relative abundance of    Firmicutes) in the cecal and/or fecal microbiota of the mammal;-   (b) measuring the F/E ratio in the cecal and/or fecal microbiota of    a healthy control;-   (c) comparing the F/E ratios measured in steps (a) and (b), and-   (d) determining that the mammal has a predisposition to the disease    if the F/E ratio is increased in the cecal and/or fecal microbiota    of the mammal as compared to the healthy control.

In a related aspect, the invention provides a method for promotingweight loss in a mammal comprising administering to the mammal atherapeutically effective amount of a probiotic composition, whereinsaid probiotic composition lowers the populations of Firmicutes in thececal and/or fecal microbiota of the mammal.

In another related embodiment, the invention provides a method forpreventing or treating a disease in a mammal selected from the groupconsisting of obesity, metabolic syndrome, diabetes mellitus,insulin-deficiency related disorders, insulin-resistance relateddisorders, glucose intolerance, non-alcoholic fatty liver, abnormallipid metabolism, and atherosclerosis, said method comprisingadministering to the mammal a therapeutically effective amount of aprobiotic composition, wherein said probiotic composition lowers thepopulations of Firmicutes in the cecal and/or fecal microbiota of themammal.

In a further embodiment, the invention provides a method for promotingweight loss in a mammal comprising administering to the mammal atherapeutically effective amount of a prebiotic composition, whereinsaid prebiotic composition lowers the populations of Firmicutes in thececal and/or fecal microbiota of the mammal.

In yet another embodiment, the invention provides a method forpreventing or treating a disease in a mammal selected from the groupconsisting of obesity, metabolic syndrome, diabetes mellitus,insulin-deficiency related disorders, insulin-resistance relateddisorders, glucose intolerance, non-alcoholic fatty liver, abnormallipid metabolism, and atherosclerosis, said method comprisingadministering to the mammal a therapeutically effective amount of aprebiotic composition, wherein said prebiotic composition lowers thepopulations of Firmicutes in the cecal and/or fecal microbiota of themammal.

In a separate embodiment, the invention provides a method of loweringthe populations of Firmicutes in the cecal and/or fecal microbiota of amammal comprising administering to the mammal a prebiotic composition.

In another embodiment, the invention provides a method for promotingweight loss in a mammal comprising administering to the mammal atherapeutically effective amount of a probiotic composition, whereinsaid probiotic composition lowers the ratio of the populations ofFirmicutes to Eubacteria (F/E ratio=relative abundance of Firmicutes) inthe cecal and/or fecal microbiota of the mammal.

In yet another embodiment, the invention provides a method forpreventing or treating a disease in a mammal selected from the groupconsisting of obesity, metabolic syndrome, diabetes mellitus,insulin-deficiency related disorders, insulin-resistance relateddisorders, glucose intolerance, non-alcoholic fatty liver, abnormallipid metabolism, and atherosclerosis, said method comprisingadministering to the mammal a therapeutically effective amount of aprobiotic composition, wherein said probiotic composition lowers theratio of the populations of Firmicutes to Eubacteria (F/E ratio=relativeabundance of Firmicutes) in the cecal and/or fecal microbiota of themammal.

In a further embodiment, the invention provides a method for promotingweight loss in a mammal comprising administering to the mammal atherapeutically effective amount of a prebiotic composition, whereinsaid prebiotic composition lowers the ratio of the populations ofFirmicutes to Eubacteria (F/E ratio=relative abundance of Firmicutes) inthe cecal and/or fecal microbiota of the mammal.

In an additional embodiment, the invention provides a method forpreventing or treating a disease in a mammal selected from the groupconsisting of obesity, metabolic syndrome, diabetes mellitus,insulin-deficiency related disorders, insulin-resistance relateddisorders, glucose intolerance, non-alcoholic fatty liver, abnormallipid metabolism, and atherosclerosis, said method comprisingadministering to the mammal a therapeutically effective amount of aprebiotic composition, wherein said prebiotic composition lowers theratio of the populations of Firmicutes to Eubacteria (F/E ratio=relativeabundance of Firmicutes) in the cecal and/or fecal microbiota of themammal.

In a separate embodiment, the invention provides a method of loweringthe ratio of the populations of Firmicutes to Eubacteria (F/Eratio=relative abundance of Firmicutes) in the cecal and/or fecalmicrobiota of the mammal comprising administering to the mammal aprebiotic composition.

In another aspect, the invention provides a method for diagnosingpredisposition to a disease in a mammal selected from the groupconsisting of obesity, metabolic syndrome, diabetes mellitus,insulin-deficiency related disorders, insulin-resistance relateddisorders, glucose intolerance, non-alcoholic fatty liver, abnormallipid metabolism, and atherosclerosis, said method comprising

-   (a) measuring the populations of at least one genus selected from    the group consisting of Coprobacillus, Sporacetigenium, Holdemania,    Dorea, Blautia, Enterococcus, Erysipelotrichaceae Incertae Sedis    (EIS), Clostridium cocleatum, and Peptosteptococcaceae IS (PIS) in    the intestinal microbiota of the mammal;-   (b) measuring the populations of the same genus in the intestinal    microbiota of a healthy control;-   (c) comparing the populations measured in steps (a) and (b), and-   (d) determining that the mammal has a predisposition to the disease    if the populations of at least one genus selected from the group    consisting of Coprobacillus, Sporacetigenium, Holdemania, Dorea,    Blautia, Enterococcus, Erysipelotrichaceae Incertae Sedis (EIS),    Clostridium cocleatum, and Peptosteptococcaceae Incertae Sedis (PIS)    in the intestinal microbiota of the mammal are decreased as compared    to the healthy control.

In a related aspect, the invention provides a method for promotingweight loss in a mammal comprising administering to the mammal atherapeutically effective amount of a probiotic composition, whereinsaid probiotic composition stimulates growth or metabolic activity of atleast one strain from the genus selected from the group consisting ofCoprobacillus, Sporacetigenium, Holdemania, Dorea, Blautia,Enterococcus, Erysipelotrichaceae Incertae Sedis (EIS), Clostridiumcocleatum, and Peptosteptococcaceae Incertae Sedis (PIS) in theintestinal microbiota of the mammal.

In a further embodiment, the invention provides a method for promotingweight loss in a mammal comprising administering to the mammal atherapeutically effective amount of a prebiotic composition, whereinsaid prebiotic composition stimulates growth or metabolic activity of atleast one strain from the genus selected from the group consisting ofCoprobacillus, Sporacetigenium, Holdemania, Dorea, Blautia,Enterococcus, Erysipelotrichaceae Incertae Sedis (EIS), Clostridiumcocleatum, and Peptosteptococcaceae Incertae Sedis (PIS) in theintestinal microbiota of the mammal.

The invention also provides a method for determining whether weight losscan be achieved in a mammal by the above two methods comprising

-   (a) measuring the populations of at least one genus selected from    the group consisting of Coprobacillus, Sporacetigenium, Holdemania,    Dorea, Blautia, Enterococcus, Erysipelotrichaceae Incertae Sedis    (EIS), Clostridium cocleatum, and Peptosteptococcaceae Incertae    Sedis (PIS) in the intestinal microbiota of the mammal;-   (b) measuring the populations of the same genus in the intestinal    microbiota of a healthy control;-   (c) comparing the populations measured in steps (a) and (b), and-   (d) determining that weight loss can be achieved in the mammal by    the above two methods if the populations of at least one genus    selected from the group consisting of Coprobacillus, Sporacetigenium    Holdemania, Dorea, Blautia, Enterococcus, Erysipelotrichaceae    Incertae Sedis (EIS), Clostridium cocleatum, and    Peptosteptococcaceae Incertae Sedis (PIS) in the intestinal    microbiota of the mammal is decreased as compared to the healthy    control.

In another related aspect, the invention provides a method forpreventing or treating a disease in a mammal selected from the groupconsisting of obesity, metabolic syndrome, diabetes mellitus,insulin-deficiency related disorders, insulin-resistance relateddisorders, glucose intolerance, non-alcoholic fatty liver, abnormallipid metabolism, and atherosclerosis, said method comprisingadministering to the mammal a therapeutically effective amount of aprobiotic composition, wherein said probiotic composition stimulatesgrowth or metabolic activity of at least one strain from the genusselected from the group consisting of Coprobacillus, Sporacetigenium,Holdemania, Dorea, Blautia, Enterococcus, Erysipelotrichaceae IncertaeSedis (EIS), Clostridium cocleatum, and Peptosteptococcaceae IncertaeSedis (PIS) in the intestinal microbiota of the mammal.

In yet another embodiment, the invention provides a method forpreventing or treating a disease in a mammal selected from the groupconsisting of obesity, metabolic syndrome, diabetes mellitus,insulin-deficiency related disorders, insulin-resistance relateddisorders, glucose intolerance, non-alcoholic fatty liver, abnormallipid metabolism, and atherosclerosis, said method comprisingadministering to the mammal a therapeutically effective amount of aprebiotic composition, wherein said prebiotic composition stimulatesgrowth or metabolic activity of at least one strain from the genusselected from the group consisting of Coprobacillus, Sporacetigenium,Holdemania, Dorea, Blautia, Enterococcus, Erysipelotrichaceae IncertaeSedis (EIS), Clostridium cocleatum, and Peptosteptococcaceae IncertaeSedis (PIS) in the intestinal microbiota of the mammal.

The invention also provides a method for determining whether a diseasein a mammal selected from the group consisting of obesity, metabolicsyndrome, diabetes mellitus, insulin-deficiency related disorders,insulin-resistance related disorders, glucose intolerance, non-alcoholicfatty liver, abnormal lipid metabolism, and atherosclerosis can beprevented or treated by the above two methods comprising

-   (a) measuring the populations of at least one genus selected from    the group consisting of Coprobacillus, Sporacetigenium, Holdemania,    Dorea, Blautia, Enterococcus, Erysipelotrichaceae Incertae Sedis    (EIS), Clostridium cocleatum, and Peptosteptococcaceae Incertae    Sedis (PIS) in the intestinal microbiota of the mammal;-   (b) measuring the populations of the same genus in the intestinal    microbiota of a healthy control;-   (c) comparing the populations measured in steps (a) and (b), and-   (d) determining that the disease in the mammal can be prevented or    treated by the above two methods if the populations of at least one    genus selected from the group consisting of Coprobacillus,    Sporacetigenium, Holdemania, Dorea, Blautia, Enterococcus,    Erysipelotrichaceae Incertae Sedis (EIS), Clostridium cocleatum, and    Peptosteptococcaceae Incertae Sedis (PIS) in the intestinal    microbiota of the mammal is decreased as compared to the healthy    control.

In a separate embodiment, the invention provides a method of stimulatinggrowth or metabolic activity of at least one strain from the genusselected from the group consisting of Coprobacillus, Sporacetigenium,Holdemania, Dorea, Blautia, Enterococcus, Erysipelotrichaceae IncertaeSedis (EIS), Clostridium cocleatum, and Peptosteptococcaceae IncertaeSedis (PIS) in the intestinal microbiota of a mammal comprisingadministering to the mammal a prebiotic composition.

In another aspect, the invention provides a method for diagnosingpredisposition to a disease in a mammal selected from the groupconsisting of obesity, metabolic syndrome, diabetes mellitus,insulin-deficiency related disorders, insulin-resistance relateddisorders, glucose intolerance, non-alcoholic fatty liver, abnormallipid metabolism, and atherosclerosis, said method comprising

-   (a) measuring the populations of at least one taxon selected from    the group consisting of Johnsonella, Oscillibacter, Lachnospiraceae,    Ruminococcaceae, and Clostridiales in the intestinal microbiota of    the mammal;-   (b) measuring the populations of the same taxonin the intestinal    microbiota of a healthy control;-   (c) comparing the populations measured in steps (a) and (b), and-   (d) determining that the mammal has a predisposition to the disease    if the populations of at least one taxon selected from the group    consisting of Johnsonella, Oscillibacter, Lachnospiraceae,    Ruminococcaceae, and Clostridiales in the intestinal microbiota of    the mammal is increased as compared to the healthy control.

In a related aspect, the invention provides a method for promotingweight loss in a mammal comprising administering to the mammal atherapeutically effective amount of a composition or a compound, whereinsaid composition or compound inhibits growth or metabolic activity of atleast one strain from the taxon selected from the group consisting ofJohnsonella, Oscillibacter, Lachnospiraceae, Ruminococcaceae, andClostridiales in the intestinal microbiota of the mammal.

In another aspect, the invention provides a method for preventing ortreating a disease in a mammal selected from the group consisting ofobesity, metabolic syndrome, diabetes mellitus, insulin-deficiencyrelated disorders, insulin-resistance related disorders, glucoseintolerance, non-alcoholic fatty liver, abnormal lipid metabolism, andatherosclerosis, said method comprising administering to the mammal atherapeutically effective amount of a composition or a compound, whereinsaid composition or compound inhibits growth or activity of at least onestrain from the taxon selected from the group consisting of Johnsonella,Oscillibacter, Lachnospiraceae, Ruminococcaceae, and Clostridiales inthe intestinal microbiota of the mammal.

In one specific embodiment, the compound used in the above two methodsis a narrow spectrum antibiotic. In another specific embodiment, thecomposition used in the above two methods is a probiotic compositioncomprising at least one strain from the genus selected from the groupconsisting of Coprobacillus, Sporacetigenium, Holdemania, Dorea,Blautia, Enterococcus, Erysipelotrichaceae Incertae Sedis (EIS),Clostridium cocleatum, and Peptosteptococcaceae Incertae Sedis (PIS).

In a related embodiment, the invention provides a method for determiningwhether weight loss can be achieved in a mammal by the above methodcomprising

-   (a) measuring the populations of at least one taxon selected from    the group consisting of Johnsonella, Oscillibacter, Lachnospiraceae,    Ruminococcaceae, and Clostridiales in the intestinal microbiota of    the mammal;-   (b) measuring the populations of the same taxon in the intestinal    microbiota of a healthy control;-   (c) comparing the populations measured in steps (a) and (b), and-   (d) determining that weight loss can be achieved in the mammal by    the above method if the populations of at least one taxon selected    from the group consisting of Johnsonella, Oscillibacter,    Lachnospiraceae, Ruminococcaceae, and Clostridiales in the    intestinal microbiota of the mammal is increased as compared to the    healthy control.

In another related embodiment, the invention provides a method fordetermining whether a disease in a mammal selected from the groupconsisting of obesity, metabolic syndrome, diabetes mellitus,insulin-deficiency related disorders, insulin-resistance relateddisorders, glucose intolerance, non-alcoholic fatty liver, abnormallipid metabolism, and atherosclerosis can be prevented or treated by theabove method comprising

-   (a) measuring the populations of at least one taxon selected from    the group consisting of Johnsonella, Oscillibacter, Lachnospiraceae,    Ruminococcaceae, and Clostridiales in the intestinal microbiota of    the mammal;-   (b) measuring the populations of the same taxon in the intestinal    microbiota of a healthy control;-   (c) comparing the populations measured in steps (a) and (b), and-   (d) determining that the disease in the mammal can be prevented or    treated by the above method if the populations of at least one taxon    selected from the group consisting of Johnsonella, Oscillibacter,    Lachnospiraceae, Ruminococcaceae, and Clostridiales in the    intestinal microbiota of the mammal is increased as compared to the    healthy control.

In a separate embodiment, the invention provides a method of inhibitinggrowth or metabolic activity of at least one strain from the taxonselected from the group consisting of Johnsonella, Oscillibacter,Lachnospiraceae, Ruminococcaceae, and Clostridiales in the intestinalmicrobiota of a mammal comprising administering to the mammal aprebiotic composition.

In another aspect, the invention provides a method for diagnosingpredisposition to a disease in a mammal selected from the groupconsisting of obesity, metabolic syndrome, diabetes mellitus,insulin-deficiency related disorders, insulin-resistance relateddisorders, glucose intolerance, non-alcoholic fatty liver, abnormallipid metabolism, and atherosclerosis, said method comprising

-   (a) measuring the populations of at least one genus selected from    the group consisting of Coprobacillus (C), Sporacetigenium (S), and    Holdemania (H), in the intestinal microbiota of the mammal;-   (b) measuring the populations of at least one genus selected from    Johnsonella (J) and Oscillibacter (O) in the intestinal microbiota    of the mammal;-   (c) determining a ratio of one of C+S+H, C+H, C+S, S+H, C, S, or H    as measured in step (a) to one of J+O, J, or O as measured in step    (b), and-   (d) determining that the mammal has a predisposition to the disease    if the ratio in step (c) is below 1, or determining that the mammal    has no predisposition to the disease if the ratio in step (c) is    above 3.

In a related aspect, the invention provides a method for promotingweight loss in a mammal comprising administering to the mammal atherapeutically effective amount of a probiotic composition, whereinsaid probiotic composition increases the ratio of populations of one of(a) Coprobacillus (C), Sporacetigenium (S), Holdemania (H), C+H, C+S,S+H, or C+S+H to populations of one of (b) Johnsonella (J),Oscillibacter (O), or J+O to above 3 in the intestinal microbiota of themammal.

In another embodiment, the invention provides a method for preventing ortreating a disease in a mammal selected from the group consisting ofobesity, metabolic syndrome, diabetes mellitus, insulin-deficiencyrelated disorders, insulin-resistance related disorders, glucoseintolerance, non-alcoholic fatty liver, abnormal lipid metabolism, andatherosclerosis, said method comprising administering to the mammal atherapeutically effective amount of a probiotic composition, whereinsaid probiotic composition increases the ratio of populations of one of(a) Coprobacillus (C), Sporacetigenium (S), Holdemania (H), C+H, C+S,S+H, or C+S+H to populations of one of (b) Johnsonella (J),Oscillibacter (O), or J+O to above 3 in the intestinal microbiota of themammal.

In a further embodiment, the invention provides a method for promotingweight loss in a mammal comprising administering to the mammal atherapeutically effective amount of a prebiotic composition, whereinsaid prebiotic composition increases the ratio of populations of one of(a) Coprobacillus (C), Sporacetigenium (S), Holdemania (H), C+H, C+S,S+H, or C+S+H to populations of one of (b) Johnsonella (J),Oscillibacter (O), or J+O to above 3 in the intestinal microbiota of themammal.

In yet another embodiment, the invention provides a method forpreventing or treating a disease in a mammal selected from the groupconsisting of obesity, metabolic syndrome, diabetes mellitus,insulin-deficiency related disorders, insulin-resistance relateddisorders, glucose intolerance, non-alcoholic fatty liver, abnormallipid metabolism, and atherosclerosis, said method comprisingadministering to the mammal a therapeutically effective amount of aprebiotic composition, wherein said prebiotic composition increases theratio of populations of one of (a) Coprobacillus (C), Sporacetigenium(S), Holdemania (H), C+H, C+S, S+H, or C+S+H to populations of one of(b) Johnsonella (J), Oscillibacter (O), or J+O to above 3 in theintestinal microbiota of the mammal.

In a separate embodiment, the invention provides a method of increasingthe ratio of populations of one of (a) Coprobacillus (C),Sporacetigenium (S), Holdemania (H), C+H, C+S, S+H, or C+S+H topopulations of one of (b) Johnsonella (J), Oscillibacter (O), or J+O inthe intestinal microbiota of a mammal comprising administering to themammal a prebiotic composition.

In another aspect, the invention provides a method for diagnosingpredisposition to a disease in a mammal selected from the groupconsisting of obesity, metabolic syndrome, diabetes mellitus,insulin-deficiency related disorders, insulin-resistance relateddisorders, glucose intolerance, non-alcoholic fatty liver, abnormallipid metabolism, and atherosclerosis, said method comprising

-   (a) measuring the populations of at least one genus selected from    the group consisting of Erysipelotrichaceae Incertae Sedis (EIS),    Peptostreptococcaceae Incertae Sedis (PIS), and Clostridium    cocleatum (Cc) in the intestinal microbiota of the mammal;-   (b) measuring the populations of Johnsonella (J) in the intestinal    microbiota of the mammal;-   (c) determining a ratio of one of EIS, PIS, EIS+PIS, or Cc as    measured in step (a) to J as measured in step (b), and-   (d) determining that the mammal has a predisposition to the disease    if the ratio in step (c) is below 1, or determining that the mammal    has no predisposition to the disease if the ratio in step (c) is    above 1.

In a related aspect, the invention provides a method for promotingweight loss in a mammal comprising administering to the mammal atherapeutically effective amount of a probiotic composition, whereinsaid probiotic composition increases the ratio of populations of one of(a) Erysipelotrichaceae Incertae Sedis (EIS), PeptostreptococcaceaeIncertae Sedis (PIS), Clostridium cocleatum (Cc), or EIS+PIS topopulations of (b) Johnsonella (J) to above 1 in the intestinalmicrobiota of the mammal.

In another embodiment, the invention provides a method for preventing ortreating a disease in a mammal selected from the group consisting ofobesity, metabolic syndrome, diabetes mellitus, insulin-deficiencyrelated disorders, insulin-resistance related disorders, glucoseintolerance, non-alcoholic fatty liver, abnormal lipid metabolism, andatherosclerosis, said method comprising administering to the mammal atherapeutically effective amount of a probiotic composition, whereinsaid probiotic composition increases the ratio of populations of one of(a) Erysipelotrichaceae Incertae Sedis (EIS), PeptostreptococcaceaeIncertae Sedis (PIS), Clostridium cocleatum (Cc), or EIS+PIS topopulations of (b) Johnsonella (J) to above 1 in the intestinalmicrobiota of the mammal.

In a further embodiment, the invention provides a method for promotingweight loss in a mammal comprising administering to the mammal atherapeutically effective amount of a prebiotic composition, whereinsaid prebiotic composition increases the ratio of populations of one of(a) Erysipelotrichaceae Incertae Sedis (EIS), PeptostreptococcaceaeIncertae Sedis (PIS), Clostridium cocleatum (Cc), or EIS+PIS topopulations of (b) Johnsonella (J) to above 1 in the intestinalmicrobiota of the mammal.

In yet another embodiment, the invention provides a method forpreventing or treating a disease in a mammal selected from the groupconsisting of obesity, metabolic syndrome, diabetes mellitus,insulin-deficiency related disorders, insulin-resistance relateddisorders, glucose intolerance, non-alcoholic fatty liver, abnormallipid metabolism, and atherosclerosis, said method comprisingadministering to the mammal a therapeutically effective amount of aprebiotic composition, wherein said prebiotic composition increases theratio of populations of one of (a) Erysipelotrichaceae Incertae Sedis(EIS), Peptostreptococcaceae Incertae Sedis (PIS), Clostridium cocleatum(Cc), or EIS+PIS to populations of (b) Johnsonella (J) to above 1 in theintestinal microbiota of the mammal.

In a separate embodiment, the invention provides a method of increasingthe ratio of populations of one of (a) Erysipelotrichaceae IncertaeSedis (EIS), Peptostreptococcaceae Incertae Sedis (PIS), Clostridiumcocleatum (Cc), or EIS+PIS to populations of (b) Johnsonella (J) in theintestinal microbiota of a mammal comprising administering to the mammala prebiotic composition.

In an additional embodiment, the invention provides a method fordiagnosing predisposition to a disease in a mammal selected from thegroup consisting of obesity, metabolic syndrome, diabetes mellitus,insulin-deficiency related disorders, insulin-resistance relateddisorders, glucose intolerance, non-alcoholic fatty liver, abnormallipid metabolism, and atherosclerosis, said method comprising

-   (a) measuring the populations of at least one genus selected from    the group consisting of Coprobacillus (C), Sporacetigenium (S),    Holdemania (H), Erysipelotrichaceae Incertae Sedis (EIS),    Peptostreptococcaceae Incertae Sedis (PIS), and Clostridium    cocleatum (Cc) in the intestinal microbiota of the mammal;-   (b) measuring the populations of Firmicutes (F) in the intestinal    microbiota of the mammal;-   (c) determining a ratio of one of C+S+H, C+H, C+S, S+H, C, S, H,    EIS, PIS, or Cc as measured in step (a) to F as measured in step    (b), and-   (d) determining that the mammal has a predisposition to the disease    if the ratio in step (c) is below 0.1, or determining that the    mammal has no predisposition to the disease if the ratio in step (c)    is above 0.1.

In a related embodiment, the invention provides a method for promotingweight loss in a mammal comprising administering to the mammal atherapeutically effective amount of a probiotic composition, whereinsaid probiotic composition increases the ratio of populations of one of(a) Coprobacillus (C), Sporacetigenium (S), Holdemania (H),Erysipelotrichaceae Incertae Sedis (EIS), Peptostreptococcaceae IncertaeSedis (PIS), Clostridium cocleatum (Cc), C+H, C+S, S+H, or C+S+H topopulations of (b) Firmicutes (F) to above 0.1 in the intestinalmicrobiota of the mammal.

In another embodiment, the invention provides a method for preventing ortreating a disease in a mammal selected from the group consisting ofobesity, metabolic syndrome, diabetes mellitus, insulin-deficiencyrelated disorders, insulin-resistance related disorders, glucoseintolerance, non-alcoholic fatty liver, abnormal lipid metabolism, andatherosclerosis, said method comprising administering to the mammal atherapeutically effective amount of a probiotic composition, whereinsaid probiotic composition increases the ratio of populations of one of(a) Coprobacillus (C), Sporacetigenium (S), Holdemania (H),Erysipelotrichaceae Incertae Sedis (EIS), Peptostreptococcaceae IncertaeSedis (PIS), Clostridium cocleatum (Cc), C+H, C+S, S+H, or C+S+H topopulations of (b) Firmicutes (F) to above 0.1 in the intestinalmicrobiota of the mammal.

In yet another embodiment, the invention provides a method for promotingweight loss in a mammal comprising administering to the mammal atherapeutically effective amount of a prebiotic composition, whereinsaid prebiotic composition increases the ratio of populations of one of(a) Coprobacillus (C), Sporacetigenium (S), Holdemania (H),Erysipelotrichaceae Incertae Sedis (EIS), Peptostreptococcaceae IncertaeSedis (PIS), Clostridium cocleatum (Cc), C+H, C+S, S+H, or C+S+H topopulations of (b) Firmicutes (F) to above 0.1 in the intestinalmicrobiota of the mammal.

In a further embodiment, the invention provides a method for preventingor treating a disease in a mammal selected from the group consisting ofobesity, metabolic syndrome, diabetes mellitus, insulin-deficiencyrelated disorders, insulin-resistance related disorders, glucoseintolerance, non-alcoholic fatty liver, abnormal lipid metabolism, andatherosclerosis, said method comprising administering to the mammal atherapeutically effective amount of a prebiotic composition, whereinsaid prebiotic composition increases the ratio of populations of one of(a) Coprobacillus (C), Sporacetigenium (S), Holdemania (H),Erysipelotrichaceae Incertae Sedis (EIS), Peptostreptococcaceae IncertaeSedis (PIS), Clostridium cocleatum (Cc), C+H, C+S, S+H, or C+S+H topopulations of (b) Firmicutes (F) to above 0.1 in the intestinalmicrobiota of the mammal.

In a separate embodiment, the invention provides a method of increasingthe ratio of populations of one of (a) Coprobacillus (C),Sporacetigenium (S), Holdemania (H), Erysipelotrichaceae Incertae Sedis(EIS), Peptostreptococcaceae Incertae Sedis (PIS), Clostridium cocleatum(Cc), C+H, C+S, S+H, or C+S+H to populations of (b) Firmicutes (F) inthe intestinal microbiota of a mammal comprising administering to themammal a prebiotic composition.

In another aspect, the invention provides a method for diagnosingpredisposition to a disease in a mammal selected from the groupconsisting of obesity, metabolic syndrome, diabetes mellitus,insulin-deficiency related disorders, insulin-resistance relateddisorders, glucose intolerance, non-alcoholic fatty liver, abnormallipid metabolism, and atherosclerosis, said method comprising

-   (a) measuring the populations of at least one family selected from    Erysipelotrichaceae and Peptostreptococcacea, in the intestinal    microbiota of the mammal;-   (b) measuring the populations of at least one family selected from    Lachnospiraceae and Ruminococcaceae in the intestinal microbiota of    the mammal;-   (c) determining a ratio of one of    Erysipelotrichaceae+Peptostreptococcacea, Erysipelotrichaceae, or    Peptostreptococcacea as measured in step (a) to one of    Lachnospiraceae+Ruminococcaceae, Lachnospiraceae, or Ruminococcaceae    as measured in step (b), and (d) determining that the mammal has a    predisposition to the disease if the ratio in step (c) is below 0.1,    or determining that the mammal has no predisposition to the disease    if the ratio in step (c) is above 0.1.

In a related embodiment, the invention provides a method for promotingweight loss in a mammal comprising administering to the mammal atherapeutically effective amount of a probiotic composition, whereinsaid probiotic composition increases the ratio of populations of one of(a) Erysipelotrichaceae+Peptostreptococcacea, Erysipelotrichaceae, orPeptostreptococcacea to populations of one of (b)Lachnospiraceae+Ruminococcaceae, Lachnospiraceae, or Ruminococcaceae toabove 0.1 in the intestinal microbiota of the mammal.

In another embodiment, the invention provides a method for preventing ortreating a disease in a mammal selected from the group consisting ofobesity, metabolic syndrome, diabetes mellitus, insulin-deficiencyrelated disorders, insulin-resistance related disorders, glucoseintolerance, non-alcoholic fatty liver, abnormal lipid metabolism, andatherosclerosis, said method comprising administering to the mammal atherapeutically effective amount of a probiotic composition, whereinsaid probiotic composition increases the ratio of populations of one of(a) Erysipelotrichaceae+Peptostreptococcacea, Erysipelotrichaceae, orPeptostreptococcacea to populations of one of (b)Lachnospiraceae+Ruminococcaceae, Lachnospiraceae, or Ruminococcaceae toabove 0.1 in the intestinal microbiota of the mammal.

In a further embodiment, the invention provides a method for promotingweight loss in a mammal comprising administering to the mammal atherapeutically effective amount of a prebiotic composition, whereinsaid prebiotic composition increases the ratio of populations of one of(a) Erysipelotrichaceae+Peptostreptococcacea, Erysipelotrichaceae, orPeptostreptococcacea to populations of one of (b)Lachnospiraceae+Ruminococcaceae, Lachnospiraceae, or Ruminococcaceae toabove 0.1 in the intestinal microbiota of the mammal.

In yet another embodiment, the invention provides a method forpreventing or treating a disease in a mammal selected from the groupconsisting of obesity, metabolic syndrome, diabetes mellitus,insulin-deficiency related disorders, insulin-resistance relateddisorders, glucose intolerance, non-alcoholic fatty liver, abnormallipid metabolism, and atherosclerosis, said method comprisingadministering to the mammal a therapeutically effective amount of aprebiotic composition, wherein said prebiotic composition increases theratio of populations of one of (a)Erysipelotrichaceae+Peptostreptococcacea, Erysipelotrichaceae, orPeptostreptococcacea to populations of one of (b)Lachnospiraceae+Ruminococcaceae, Lachnospiraceae, or Ruminococcaceae toabove 0.1 in the intestinal microbiota of the mammal.

In a separate embodiment, the invention provides a method of increasingthe ratio of populations of one of (a)Erysipelotrichaceae+Peptostreptococcacea, Erysipelotrichaceae, orPeptostreptococcacea to populations of one of (b)Lachnospiraceae+Ruminococcaceae, Lachnospiraceae, or Ruminococcaceae inthe intestinal microbiota of a mammal comprising administering to themammal a prebiotic composition.

In any of the above methods, the populations of bacteria can bedetermined by any method known in the art. In a preferred embodiment,the populations of bacteria are determined by qPCR of bacterial 16SrRNA.

In another aspect, the invention provides a method for diagnosingpredisposition to a disease in a mammal selected from the groupconsisting of obesity, metabolic syndrome, diabetes mellitus,insulin-deficiency related disorders, insulin-resistance relateddisorders, glucose intolerance, non-alcoholic fatty liver, abnormallipid metabolism, and atherosclerosis, said method comprising

-   (a) measuring the total number of Butyryl CoA transferase    (BCoAT)-encoding genes in the intestinal microbiota of the mammal;-   (b) measuring the total number of BCoAT-encoding genes in the    intestinal microbiota of a healthy control;-   (c) comparing the total number of BCoAT-encoding genes measured in    steps (a) and (b), and-   (d) determining that the mammal has a predisposition to the disease    if the total number of BCoAT-encoding genes in the intestinal    microbiota of the mammal is increased as compared to the healthy    control.

In another aspect, the invention provides a method for diagnosingpredisposition to a disease in a mammal selected from the groupconsisting of obesity, metabolic syndrome, diabetes mellitus,insulin-deficiency related disorders, insulin-resistance relateddisorders, glucose intolerance, non-alcoholic fatty liver, abnormallipid metabolism, and atherosclerosis, said method comprising

-   (a) measuring a ratio of the total number of Butyryl CoA transferase    (BCoAT)-encoding genes to copies of Bacteroidetes 16S rRNA in the    intestinal microbiota of the mammal;-   (b) measuring a ratio of the total number of BCoAT-encoding genes to    copies of Bacteroidetes 16S rRNA in the intestinal microbiota of a    healthy control;-   (c) comparing the ratios of the total number of BCoAT-encoding genes    to copies of Bacteroidetes 16S rRNA measured in steps (a) and (b),    and-   (d) determining that the mammal has a predisposition to the disease    if the ratio of the total number of BCoAT-encoding genes to copies    of Bacteroidetes 16S rRNA in the intestinal microbiota of the mammal    is increased as compared to the healthy control.

In the above methods, the total number of BCoAT-encoding genes andcopies of Bacteroidetes 16S rRNA can be measured by any method known inthe art. In a preferred embodiment, the total number of BCoAT-encodinggenes and copies of Bacteroidetes 16S rRNA are measured by qPCR.

In a related aspect, the invention provides a method for promotingweight loss in a mammal comprising administering to the mammal atherapeutically effective amount of a probiotic composition, whereinsaid probiotic composition lowers the levels of Butyryl CoA transferase(BCoAT) enzyme and/or the levels of butyrate in the intestinalmicrobiota of the mammal.

In another embodiment, the invention provides a method for preventing ortreating a disease in a mammal selected from the group consisting ofobesity, metabolic syndrome, diabetes mellitus, insulin-deficiencyrelated disorders, insulin-resistance related disorders, glucoseintolerance, non-alcoholic fatty liver, abnormal lipid metabolism, andatherosclerosis, said method comprising administering to the mammal atherapeutically effective amount of a probiotic composition, whereinsaid probiotic composition lowers the levels of Butyryl CoA transferase(BCoAT) enzyme and/or the levels of butyrate in the intestinalmicrobiota of the mammal.

In yet another embodiment, the invention provides a method for promotingweight loss in a mammal comprising administering to the mammal atherapeutically effective amount of a prebiotic composition, whereinsaid prebiotic composition lowers the levels of Butyryl CoA transferase(BCoAT) enzyme and/or the levels of butyrate in the intestinalmicrobiota of the mammal.

In a further embodiment, the invention provides a method for preventingor treating a disease in a mammal selected from the group consisting ofobesity, metabolic syndrome, diabetes mellitus, insulin-deficiencyrelated disorders, insulin-resistance related disorders, glucoseintolerance, non-alcoholic fatty liver, abnormal lipid metabolism, andatherosclerosis, said method comprising administering to the mammal atherapeutically effective amount of a prebiotic composition, whereinsaid prebiotic composition lowers the levels of Butyryl CoA transferase(BCoAT) enzyme and/or the levels of butyrate in the intestinalmicrobiota of the mammal.

In a separate embodiment, the invention provides a method of loweringthe levels of Butyryl CoA Transferase (BCoAT) enzyme and/or the levelsof butyrate in the intestinal microbiota of a mammal comprisingadministering to the mammal a prebiotic composition.

In any of the above methods, the levels of BCoAT enzyme can be measuredby any method known in the art. In a preferred embodiment, the levels ofBCoAT enzyme are measured by determining the total number ofBCoAT-encoding genes. In another preferred embodiment, the levels ofBCoAT enzyme are measured by BCoAT enzyme functional assay.

In any of the above methods, the levels of butyrate can be measured byany method known in the art. In a preferred embodiment, the levels ofbutyrate are measured using chromatographic methods.

In another aspect, the invention provides a method for promoting weightloss in a mammal comprising administering to the mammal atherapeutically effective amount of a probiotic composition, whereinsaid probiotic composition lowers the ratio of the total number ofButyryl CoA transferase (BCoAT)-encoding genes to copies ofBacteroidetes 16S rRNA in the intestinal microbiota of the mammal.

In yet another aspect, the invention provides a method for preventing ortreating a disease in a mammal selected from the group consisting ofobesity, metabolic syndrome, diabetes mellitus, insulin-deficiencyrelated disorders, insulin-resistance related disorders, glucoseintolerance, non-alcoholic fatty liver, abnormal lipid metabolism, andatherosclerosis, said method comprising administering to the mammal atherapeutically effective amount of a probiotic composition, whereinsaid probiotic composition lowers the ratio of the total number ofButyryl CoA transferase (BCoAT)-encoding genes to copies ofBacteroidetes 16S rRNA in the intestinal microbiota of the mammal.

In a further aspect, the invention provides a method for promotingweight loss in a mammal comprising administering to the mammal atherapeutically effective amount of a prebiotic composition, whereinsaid prebiotic composition lowers the ratio of the total number ofButyryl CoA transferase (BCoAT)-encoding genes to copies ofBacteroidetes 16S rRNA in the intestinal microbiota of the mammal.

In an additional aspect, the invention provides a method for preventingor treating a disease in a mammal selected from the group consisting ofobesity, metabolic syndrome, diabetes mellitus, insulin-deficiencyrelated disorders, insulin-resistance related disorders, glucoseintolerance, non-alcoholic fatty liver, abnormal lipid metabolism, andatherosclerosis, said method comprising administering to the mammal atherapeutically effective amount of a prebiotic composition, whereinsaid prebiotic composition lowers the ratio of the total number ofButyryl CoA transferase (BCoAT)-encoding genes to copies ofBacteroidetes 16S rRNA in the intestinal microbiota of the mammal.

In a separate embodiment, the invention provides a method of loweringthe ratio of the total number of Butyryl CoA Transferase(BCoAT)-encoding genes to copies of bacteroidetes 16S rRNA in theintestinal microbiota of a mammal comprising administering to the mammala prebiotic composition.

In the above methods, the total number of BCoAT-encoding genes andcopies of Bacteroidetes 16S rRNA can be measured by any method known inthe art. In a preferred embodiment, the total number of BCoAT-encodinggenes and copies of Bacteroidetes 16S rRNA are measured by qPCR.

In conjunction with therapeutic methods, the present invention alsoprovides various probiotic and prebiotic compositions which can be usedin such methods. Probiotic compositions according to the presentinvention can contain live bacterial strains and/or spores and alsoinclude conditionally lethal bacterial strains. Non-limiting examples ofuseful bacterial strains include, e.g., strains from the generaCoprobacillus, Sporacetigenium, Holdemania, Dorea, Blautia,Enterococcus, Erysipelotrichaceae Incertae Sedis (EIS), Clostridiumcocleatum, and Peptosteptococcaceae Incertae Sedis (PIS).

Probiotic compositions of the present invention can further comprise abuffering agent such as, e.g., sodium bicarbonate, juice, milk, yogurt,infant formula, etc.

Probiotic compositions of the present invention can be administeredconjointly with a prebiotic composition which stimulates growth and/ormetabolic activity of bacteria contained in the probiotic composition.Such combinations of probiotic and prebiotic compositions can beadministered in one composition or as two separate compositions(administered simultaneously or sequentially).

In a specific embodiment, a probiotic composition further comprises acompound selected from the group consisting of xylose, arabinose,ribose, galactose, rhamnose, cellobiose, fructose, lactose, salicin,sucrose, glucose, esculin, tween 80, trehalose, maltose, mannose,mellibiose, raffinose, fructooligosaccharides, galacto-oligosaccharides,amino acids, alcohols, and any combinations thereof. In another specificembodiment, a probiotic composition further comprises a compoundselected from the group consisting of trehalose, cellobiose, maltose,mannose, sucrose, fructose, galactose, lactose, salicin, melibiose,raffinose, and any combinations thereof. In yet another specificembodiment, a probiotic composition further comprises a compoundselected from the group consisting of water-soluble cellulosederivatives, water-insoluble cellulose derivatives, unprocessed oatmeal,metamucil, all-bran, and any combinations thereof. In a preferredembodiment, the water-soluble cellulose derivative is selected from thegroup consisting of methylcellulose, methyl ethyl cellulose,hydroxyethyl cellulose, ethyl hydroxyethyl cellulose, cationichydroxyethyl cellulose, hydroxypropyl cellulose, hydroxyethylmethylcellulose, hydroxypropyl methylcellulose, and carboxymethylcellulose. In another preferred embodiment, the water-insolublecellulose derivative is ethyl cellulose.

In one specific embodiment, the invention provides a probioticcomposition comprising one or more strain from the genus Holdemania andone or more compounds selected from the group consisting of Tween 80,esculin, fructose, glucose, lactose, maltose, salicin, and sucrose.

In another specific embodiment, the invention provides a probioticcomposition comprising one or more strain from the genusSporoacetigenicum and one or more compounds selected from the groupconsisting of arabinose, fructose, glucose, maltose, and xylose.

In yet another specific embodiment, the invention provides a probioticcomposition comprising one or more strain from the genus Coprobacillusand one or more compounds selected from the group consisting of mannose,fructose, sucrose, maltose, cellobiose, trehalose, salicin, lactose,glucose, and galactose.

In yet another specific embodiment, the invention provides a probioticcomposition comprising one or more strain from the genus Clostridiumcocleatum and one or more compounds selected from the group consistingof cellobiose, fructose, galactose, glucose, inulin, lactose, maltose,mannose, mellibiose, raffinose, and sucrose.

In one embodiment, the invention provides a prebiotic composition usefulin the methods of the present invention which prebiotic compositioncomprises a compound selected from the group consisting of trehalose,cellobiose, maltose, mannose, sucrose, fructose, galactose, lactose,salicin, melibiose, raffinose, and any combinations thereof. In anotherembodiment, the invention provides a prebiotic composition comprising acompound selected from the group consisting of xylose, arabinose,ribose, galactose, rhamnose, cellobiose, fructose, lactose, salicin,sucrose, glucose, esculin, tween 80, trehalose, maltose, mannose,mellibiose, raffinose, fructooligosaccharides, galactooligosaccharides,amino acids, alcohols, water-soluble cellulose derivatives,water-insoluble cellulose derivatives, unprocessed oatmeal, metamucil,all-bran, and any combinations thereof. In one preferred embodiment, thewater-soluble cellulose derivative is selected from the group consistingof methylcellulose, methyl ethyl cellulose, hydroxyethyl cellulose,ethyl hydroxyethyl cellulose, cationic hydroxyethyl cellulose,hydroxypropyl cellulose, hydroxyethyl methylcellulose, hydroxypropylmethylcellulose, and carboxymethyl cellulose. In another preferredembodiment, the water-insoluble cellulose derivative is ethyl cellulose.

Probiotic and prebiotic compositions useful in the methods of thepresent invention can be formulated in different forms (e.g., as aliquid solution, powder, capsule, tablet, suppository, etc.) and can beadministered by various methods (e.g., orally, rectally, viaesophagogastroduodenoscopy, colonoscopy, nasogastric tube, orogastrictube, etc.).

In one embodiment, the mammal in any of the above methods is human.

In one embodiment, the mammal in any of the above methods is on a highfat diet.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D are plots showing the effect of diet on host metabolism.Adult C57BL/6 mice were fed high fat diet (HFD, 60% kcal from fat) fortwo months prior to the study, then continued on HFD, or switched toeither low fat diet (LFD, 10% kcal from fat) or to HFD with 10% HPMCsupplementation (HPMC). Panels: (FIG. 1A) weight for the mice over the4-week study; (FIG. 1B) total energy intake (kcal) for the duration ofthe 4-week experiment (bar at median); (FIG. 1C) correlation by linearregression between 4-week weight change and total energy intake; (FIG.1D) Difference (mean ±SE) between actual and predicted weight change,based on energy intake and the best fit line for LFD and HFD mice. *p<0.05, **p <0.01, ***p <0.001, Mann-Whitney U test for FIG. 1B and FIG.1D, non-zero slope for FIG. 1C.

FIGS. 2A-2T are scatter plots showing changes in murine metabolicphenotypes in response to 4 weeks of dietary intervention. Panelsrepresent fasting plasma: FIG. 2A) cholesterol, FIG. 2B) HDL, FIG. 2C)LDL, FIG. 2D) VLDL, FIG. 2E) free fatty acids, FIG. 2F) triglycerides,FIG. 2G) glucose, FIG. 2H) insulin, FIG. 21) leptin, FIG. 2J)adiponectin, FIG. 2K) liver adiposity (% lipids), FIG. 2L) livertriglycerides, FIG. 2M) fecal saturated fat, FIG. 2N) fecal unsaturatedfat, FIG. 20) fecal transunsaturated fats, FIG. 2P) fecal bile acid,FIG. 2Q) fecal sterols, FIG. 2R) fecal monoacyglycerides, FIG. 2S) fecaldiacylgycerridess, and FIG. 2T) fecal triacylglycerides. *p <0.05, **p<0.01, ***p <0.001, FDR-corrected Mann-Whitney U test.

FIGS. 3A-3H are plots of quantitative PCR analysis of the intestinalmicrobiota. Analysis of fecal, cecal, and ileal samples measuringpopulation copy number per gram of sample of FIG. 3A) total bacteria,FIG. 3B) Bactoidetes, FIG. 3C) Firmicutes, FIG. 3D) BCoAT, FIG. 3E)ratio of Bacteroidetes/Firmicutes, and relative abundance (%) of FIG.3F) Bacteroidetes, FIG. 3G) Firmicutes, FIG. 3H) BCoAT, *p<0.05, **p<0.01, ***p <0.001, Mann-Whitney U test.

FIG. 4A is a bar diagram demonstrating that HPMC exposure significantlyalters the composition of the cecal microbiome compared to micemaintained on a 60% diet or those switched to a 10% diet. Mice exposedto a 60% fat diet, then switched to a diet of 60% fat +HPMC hadsignificant changes in cecal microbiota compared to the control micemaintained on the 60% fat diet alone. Notably, there were markedincreases in populations of Coprobacillus, Sporacetigenium, Holdemania,Dorea, Enterococcus, and Blautia, as well as marked decreases inJohnsonella, and Oscillibacter. Little change in cecal microbiota wasobserved in mice switched from a 60% fat diet to a 10% fat diet,compared to the control mice maintained on a 60% fat diet. Observedchanges include an increase in Dorea, and a decrease in Coprobacillusand Papillibacter. Note that at each level of taxa, there are two scalesfor abundances.

FIG. 4B is a summary table of significant changes in the cecalmicrobiome seen in FIG. 6 at different taxonomic levels.

FIG. 5 represents scatterplots showing relative abundance ofCoprobacillus, Sporacetigenium and Holdemania in fecal, cecal, and ilealsamples from C57B6 mice on diets consisting of 60% fat 10% fat, or 60%fat+HPMC. The figure demonstrates that HPMC exposure significantlyincreases Coprobacillus, Sporacetigenium, and Holdemania abundance. Theeffects of HPMC on Coprobacillus and Holdemania are seen primarily inthe cecum and are noted in the 2 and 4 fecal specimens as well.Sporacetigenium census was higher in the ileum than in the cecum or infecal pellets for all groups. *P <0.05, **P <0.01, ***P<0.001.

FIGS. 6A-6C are scatter plots of ratios that represent diagnosticcriteria for predicting predisposition to weight gain on a high fat dietand effectiveness of fiber treatment for weight loss or weight gainprevention. C57B6 mice were maintained on a 60% fat diet for 2 months,then 1 group was switched to a 10% fat diet, another switched to a 60%fat+HPMC diet, and a 3^(rd) group was maintained on the 60% fat diet.FIG. 6A. Ratios at the genus level are calculated by dividing the sum ofany combination of Coprobacillus, Sporacetigenium, and/or Holdemania(CSH) by any combination of Johnsonella and/or Oscillibacter. A ratiobelow 1 indicates a state that is predisposed to weight gain while aratio above 3 indicates a state that has a high propensity to preventweight gain. A ratio between 1 and 3 is intermediate. FIG. 6B.Additional ratios for CSH are calculated by dividing the sum of anycombination of Coprobacillus, Sporacetigenium, and/or Holdemania by thephylum Firmicutes to measure the relative abundance. A ratio below 0.1indicates a state that is predisposed to weight gain while a ratio above0.1 indicates a state that has a high propensity to prevent weight gain.FIG. 6C. Ratios at the family level are calculated by dividing the sumof any combination of Erysipelotrichaceae and/or Peptostreptococcacea byLachnospiraceae and/or Ruminococcaceae. A ratio below 0.1 indicates astate that is predisposed to weight gain while a ratio above 0.1indicates a state that has a high propensity to prevent weight gain.

FIGS. 7A-7C are graphs showing the relative abundance (%) of454-pyrosequencing reads classified at the phylum level for FIG. 7A)Firmicutes, FIG. 7B) Bacteroidetes, and FIG. 7C) the ratio ofBacteroidetes to Firmicutes.

FIGS. 8A-8D are graphs showing the relative abundance (%) of454-pyrosequencing reads classified at the class level for FIG. 8A)Clostridia, FIG. 8B) Erysipelotrichi, FIG. 8C) Bacteroidia, and FIG. 8D)Bacilli. *P <0.05, **P <0.01, ***P<0.001 for FDR-correctedMann-Whitney-U.

FIGS. 9A-9D are graphs showing the relative abundance (%) of454-pyrosequencing reads classified at the order level for FIG. 9A)Clostridiales, FIG. 9B) Erysipelotrichales, FIG. 9C) Bacteroidales, andFIG. 9D) Lactobacillales, ***P<0.001 for FDR-corrected Mann-Whitney-U.

FIGS. 10A-10G are graphs showing the relative abundance (%) of454-pyrosequencing reads classified at the family level for FIG. 10A)Lachnospiraceae, FIG. 10B) Ruminococcaceae, FIG. 10C)Erysipelotrichaeceae, FIG. 10D) Peptostreptococcaceae, FIG. 10E)Lactobacillaceae, FIG. 10F) Porphyromonadaceae, FIG. 10G)Clostridiaceae. ***P<0.001 for FDR-corrected Mann-Whitney-U.

FIGS. 11A-11E are graphs showing the relative abundance (%) of454-pyrosequencing reads classified at the genus level for FIG. 11A)Johnsonella, FIG. 11B) Erysipelotrichaceae incertae sedis, FIG. 11C)Peptostreptococcaceae incertae sedis, FIG. 11D) Clostridium, FIG. 11E)Lactobacillus. ***P<0.001 for FDR-corrected Mann-Whitney-U.

FIG. 12 shows mean relative abundance of the combined 59 OTUs with aclosest match to Clostridium cocleatum in fecal 0 week, 2 week, 4 week,and 4-week cecal and ileal samples. *p <0.05, **p <0.01, ***p <0.001.Mann-Whitney U Test.

FIGS. 13A-13F is a scatter plot of ratios based on Qiime bioinformaticpipeline that represent diagnostic criteria for predictingpredisposition to weight gain on a high fat diet and effectiveness offiber treatment for weight loss or weight gain prevention for 4-weekcecal (FIGS. 13A, 13C, 13E) and 4-week fecal (FIGS. 13B, 13D, 13F).FIGS. 13A-13B). Ratios at the genus level are calculated by dividing thesum of any combination of Erysipelotrichaceae Incertae Sedis,Peptostreptococcaceae Incertae Sedis, and/or Clostridium cocleatum byJohnsonella. A ratio below 1 indicates a state that is predisposed toweight gain while a ratio above 1 indicates a state that has a highpropensity to prevent weight gain. FIGS. 13C-13D). Additional ratios arecalculated by dividing the sum of any combination of ErysipelotrichaceaeIncertae Sedis, Peptostreptococcaceae Incertae Sedis, and/or Clostridiumcocleatum by the phylum Firmicutes to measure the relative abundance. Aratio below 0.1 indicates a state that is predisposed to weight gainwhile a ratio above 0.1 indicates a state that has a high propensity toprevent weight gain. FIGS. 13E-13F). Ratios at the family level arecalculated by dividing the sum of any combination of Erysipelotrichaceaeand/or Peptostreptococcacea by Lachnospiraceae and/or Ruminococcaceae. Aratio below 0.1 indicates a state that is predisposed to weight gainwhile a ratio above 0.1 indicates a state that has a high propensity toprevent weight gain.

FIGS. 14A and 14B are graphs showing diversity of the bacterialpopulations in the fecal and cecal microbiota at the class level.Rarefaction curves for class richness and Shannon diversity index forevenness are shown at the class level.

FIGS. 15A and 15B demonstrate assessment of microbial diversity inrelation to treatments. Top. Rarefaction curves at the OTU level forFIG. 15A) taxonomic richness and FIG. 15B) Shannon index for evenness ofthe intestinal microbiome in fecal (week 0, 2, and 4) and cecal (week 4)microbiota, according to dietary treatment.

FIGS. 16A-16G show the effect of diet and fiber on microbial communitystructure. PCA analysis of the unweighted UniFrac distances of microbial16S rDNA sequences from the V3-5 region in fecal samples at week 0(baseline) (FIG. 16A), week 2 (FIG. 16B), and week 4 (FIG. 16C), cecalsamples at sacrifice (FIG. 16F), and ileal samples at sacrifice (FIG.16G). Unweighted UniFrac distances in LFD, HFD, and HPMC mouse fecalsamples comparing community distance from 0 weeks (FIG. 16D) and from 2weeks (FIG. 16E), *p <0.001. Three principal components were plotted byKiNG Kinetic Image, Next Generation version 2.16 with each samplerepresented as a circle.

FIGS. 17A and 17B show weighted UniFrac distance of the fecal microbiomeat the OTU level. Distance (mean ±95% CI) are shown from baseline (FIG.17A), or from week 2 (FIG. 17B). ***p <0.001. As with unweighted UniFracdistances, there were no differences for the HFD mice over the course ofthe experiment, as expected. In the HPMC mice, there were progressivedifference in the community structure at weeks 2 and 4, whereas for theLFD mice, the communities stabilized after week 2.

FIGS. 18A-18E show phylogenetic differences between treatment groups.Heat map of intestinal microbiome. Representation of relative abundanceof predominant taxa classified at the family level (columns) for 30individual mice on the three different diets (rows) in (FIG. 18A) week-0fecal, (FIG. 18B) week-2 fecal, (FIG. 18C) 4-week fecal, (FIG. 18D)cecal, and (FIG. 18E) ileal samples.

FIG. 19 shows clustering of intestinal microbiota by heat map analysis.Number of samples falling within either the top or bottom major branchfor mice fed HFD, LFD, or HPMC, P-values for _(χ) ² and Fisher's exacttest for contingency.

FIG. 20 shows associations between predominant taxa in fecal specimens.Specimens were obtained at baseline (week 0) and at weeks 2 and 4 fromthe three experimental groups of mice (LFD, HFD, and HPMC). A circleindicates that Order level taxon is present at ≧1% in all specimens andthe circle size corresponds to relative abundance. Taxa classified atthe Order level are: 1, Bacteroidales; 2, Bacteroidetes: unclassified;3, Lactobacillales; 4, Clostridiales; 5, Erysipelotrichales; 6,Firmicutes: unclassified; 7, Bacteria: unclassified. A solid lineindicates a significant (p<0.05) correlation between two Orders, whereasa dashed line is not significant (p>0.05). The numerical values indicatethe strength of the correlation and the directionality (positive ornegative).

FIGS. 21A-21F show significant relationships between taxa and hostphenotype, conditioned on dietary intervention. Metabolic parameters formice on the three different diets, LFD (triangle), HFD (square), andHPMC (circle) were examined with respect to relative abundance of taxa,and the correlation constant (R) from linear regression analysis shown.Top panels represent weight change vs. cecal Firmicutes (FIG. 21A),cecal Bacteroidetes (FIG. 21B), and cecal Erysipelotrichaceae IncertaeSedis (FIG. 21C). Bottom panels represent energy intake vs. 4 week fecalLachnospiraceae (FIG. 21D), liver free cholesterol vs. cecalPorphyromonadaceae (FIG. 21E), and fecal saturated fat vs. cecalErysipelotrichaceae (FIG. 21F). *p<0.05, **p<0.01, ***p<0.001 for anon-zero slope.

FIG. 22 is an outline of experiments directed to comparing the effect ofdiet-induced obesity (DIO) diet on control mice, mice exposed to atleast one of Coprobacillus, Sporacetigenium, Holdemania,Erysipelotrichaceae Incertae Sedis, Peptostreptococaceae Incertae Sedis,and Clostridium cocleatum (CSHEPCc) or prebiotics alone, or mice exposedto CSHEPCc and prebiotics simultaneously.

FIG. 23 is an outline of experiments directed to evaluating the effectsof prophylactic exposure to Coprobacillus, Sporacetigenium, Holdemania,Erysipelotrichaceae Incertae Sedis, Peptostreptococaceae Incertae Sedis,and Clostridium cocleatum (CSHEPCc), and/or prebiotics. In thisexperiment, the mice are exposed to nothing (controls), CSHEPCc orprebiotics alone, or CSHEPCc and prebiotics upon weaning. After 4 weeks,they are given either high fat or regular chow to see the effects of theprophylactic exposure.

FIG. 24 is an outline of experiments directed to determining the effectsof specific colonizations in germ-free mice. There are three potentialtypes of studies summarized here, comparing different control and studygroups. JO is at least one of Johnsonella and Oscillibacter; CSHEPCc isat least one of Coprobacillus, Sporacetigenium, Holdemania,Erysipelotrichaceae Incertae Sedis, Peptostreptococaceae Incertae Sedis,and Clostridium cocleatum.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on an unexpected experimental observationthat prevention of weight gain associated with adding HPMC to a high-fatdiet in mice is associated with changes in the population size andcomposition of an intestinal microbiota with (i) reductions in totalbacterial populations, primarily reflecting reduction in phylumFirmicutes (the effect being significant in both the cecum and theileum), (ii) significant decreases in the populations of generaJohnsonella and Oscillibacter, family Lachnospiraceae andRuminococcaceae, order Clostridiales, class Clostridia, and phylumFirmicutes, (iii) marked increases in the populations of generaCoprobacillus, Sporacetigenium, Holdemania, Erysipelotrichaceae IncertaeSedis (EIS), Clostridium cocleatum, and Peptosteptococcaceae IncertaeSedis (PIS), moderate increases in genera Dorea, Blautia, andEnterococcus, increases in family Erysipelotrichaceae,Peptostreptococcaceae, Clostridiales Insertae Sedis XIV, andEnterococcaceae, order Erysipelotrichales, and Lactobacillales, andclass Erysipelotrichi and Bacilli, especially in relation to the totalnumbers of Firmicutes, and (iv) significant decreases in BCoAT genelevels, in a manner predicted to lower butyrate availability and energyproduction. The present invention is further based on a surprisingobservation that cellulose ethers with a beta 1,4 linkage of anhydrousglucose units have a prebiotic effect although they are known to besubstantially non-fermentable and non-digestible materials in thedigestive tract of mammals.

The present invention provides novel probiotic and prebioticcompositions and methods for diagnosing predisposition to and methodsfor treating obesity, metabolic syndrome, insulin-deficiency orinsulin-resistance related disorders, glucose intolerance, diabetesmellitus, non-alcoholic fatty liver, abnormal lipid metabolism,atherosclerosis, and related disorders based on the above-identifiedchanges in mammalian bacterial intestinal microbiota.

Definitions and Abbreviations

The term “Eubacteria” refers to all bacteria and excludes archaea. Inmammals, >90% of all colonic bacteria are in the phyla Firmicutes orBacteroidetes (Ley et al., Nat Rev Microbiol 2008; 6:776-88).

The term “intestinal microbiota” refer to bacteria in the digestivetract.

The term “cecal microbiota” refers to microbiota derived from cecum,which in mammals is the beginning region of the large intestine in theform of a pouch connecting the ileum with the ascending colon of thelarge intestine; it is separated from the ileum by the ileocecal valve(ICV), and joins the colon at the cecocolic junction.

The term “ileal microbiota” refers to microbiota derived from ileum,which in mammals is the final section of the small intestine and followsthe duodenum and jejunum; ileum is separated from the cecum by theileocecal valve (ICV).

As used herein, the term “probiotic” refers to a substantially purebacteria (i.e., a single isolate), or a mixture of desired bacteria, andmay also include any additional components that can be administered to amammal for restoring microbiota. Such compositions are also referred toherein as a “bacterial inoculant.” Probiotics or bacterial inoculantcompositions of the invention are preferably administered with abuffering agent to allow the bacteria to survive in the acidicenvironment of the stomach, i.e., to resist low pH and to grow in theintestinal environment. Such buffering agents include sodiumbicarbonate, juice, milk, yogurt, infant formula, and other dairyproducts.

As used herein, the term “prebiotic” refers to an agent that increasesthe number of one or more desired bacteria and/or desired metabolicactivity. The term “metabolic activity of bacteria” broadly refers toany aspect of microbial catabolism and/or anabolism (including, e.g.,the breakdown of carbohydrates, proteins, and lipids, secretion of smallmolecules such as, e.g., short chain fatty acids and proteins, synthesisor modifications of large molecular weight bioactive molecules thatinvolve energy generation, building of cell walls, capsules, andinternal structures) as well as to any pathway affecting the ability ofbacteria to move and/or reproduce. The metabolic activity need notrelate to the desired bacteria but can be general (e.g., BCoATactivity).

Non-limiting examples of prebiotics useful in the methods of the presentinvention include xylose, arabinose, ribose, galactose, rhamnose,cellobiose, fructose, lactose, salicin, sucrose, glucose, esculin, tween80 (e.g., 0.2%), trehalose, maltose, mannose, mellibiose, raffinose,fructooligosaccharides (e.g., oligofructose, inulin, inulin-typefructans), galactooligosaccharides, amino acids, alcohols, water-solublecellulose derivatives (most preferably, methylcellulose, methyl ethylcellulose, hydroxyethyl cellulose, ethyl hydroxyethyl cellulose,cationic hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxyethylmethylcellulose, hydroxypropyl methylcellulose, and carboxymethylcellulose), water-insoluble cellulose derivatives (most preferably,ethyl cellulose), unprocessed oatmeal, metamucil, all-bran, and anycombinations thereof. See, e.g., Ramirez-Farias et al., Br J Nutr (2008)4:1-10; Pool-Zobel and Sauer, J Nutr (2007), 137:2580S-2584S.

The term “water-soluble cellulose derivative” as used herein means thatthe cellulose derivative has a solubility in water of at least 2 grams,preferably at least 3 grams, more preferably at least 5 grams in 100grams of distilled water at 25° C. and 1 atmosphere. The term“water-soluble cellulose derivative” does not include unmodifiedcellulose itself which tends to be water-insoluble.

The term “water-insoluble cellulose derivative” as used herein does notinclude unmodified cellulose and means that the cellulose derivative hasa solubility in water of less than 2 grams, preferably less than 1 gram,in 100 grams of distilled water at 25° C. and 1 atmosphere.

The terms “treat” or “treatment” of a state, disorder or conditioninclude:

(1) preventing or delaying the appearance of at least one clinical orsub-clinical symptom of the state, disorder or condition developing in asubject that may be afflicted with or predisposed to the state, disorderor condition but does not yet experience or display clinical orsubclinical symptoms of the state, disorder or condition; or

(2) inhibiting the state, disorder or condition, i.e., arresting,reducing or delaying the development of the disease or a relapse thereof(in case of maintenance treatment) or at least one clinical orsub-clinical symptom thereof; or

(3) relieving the disease, i.e., causing regression of the state,disorder or condition or at least one of its clinical or sub-clinicalsymptoms.

The benefit to a subject to be treated is either statisticallysignificant or at least perceptible to the patient or to the physician.

A “therapeutically effective amount” means the amount of a bacterialinoculant or a compound (e.g., a prebiotic or a narrow spectrumantibiotic or anti-bacterial agent) that, when administered to a subjectfor treating a state, disorder or condition, is sufficient to effectsuch treatment. The “therapeutically effective amount” will varydepending on the compound, bacteria or analogue administered as well asthe disease and its severity and the age, weight, physical condition andresponsiveness of the subject to be treated.

The term “narrow spectrum antibiotic” is an antibiotic which canselectively inhibit growth and/or activity of one or few bacterialspecies or taxa.

The terms “diet-induced obesity (DIO) diet” and “high fat diet” are usedherein interchangeably to refer to a high-fat diet, typically of 45% or60% in total fat content, that leads to obesity, hyperglycemia,hyperinsulinemia, and hypertension in a mouse model. The composition ofthe diet was designed to approximate the typical Western diet. See,e.g., Surwit et al., Metabolism, 1995, 44:645-651.

The term “Butyryl CoA transferase (BCoAT)-encoding genes” as used hereinrefers to genes encoding an enzyme involved in the regulation ofmetabolism of short chain fatty acids and, preferably, butyratesynthesis.

As used herein, the term “metagenome” refers to genomic materialobtained directly from a subject, instead of from culture. Metagenome isthus composed of microbial and host components.

As used herein, the phrase “pharmaceutically acceptable” refers tomolecular entities and compositions that are generally regarded asphysiologically tolerable.

As used herein, the term “combination” of a bacterial inoculant,probiotic, analogue, or prebiotic compound and at least a secondpharmaceutically active ingredient means at least two, but any desiredcombination of compounds can be delivered simultaneously or sequentially(preferably, within a 24 hour period).

Within the meaning of the present invention, the term “conjointadministration” is used to refer to administration of a probiotic and aprebiotic simultaneously in one composition, or simultaneously indifferent compositions, or sequentially (preferably, within a 24 hourperiod).

“Patient” or “subject” as used herein refers to all mammals and includeshuman and veterinary animals. The term “healthy control” refers to amammal of the same species (and preferably same sex and age group) whichdoes not have a disease or condition that is being treated.

The term “carrier” refers to a diluent, adjuvant, excipient, or vehiclewith which the compound is administered. Such pharmaceutical carrierscan be sterile liquids, such as water and oils, including those ofpetroleum, animal, vegetable or synthetic origin, such as peanut oil,soybean oil, mineral oil, sesame oil and the like. Water or aqueoussolution saline solutions and aqueous dextrose and glycerol solutionsare preferably employed as carriers, particularly for injectablesolutions. Alternatively, the carrier can be a solid dosage formcarrier, including but not limited to one or more of a binder (forcompressed pills), a glidant, an encapsulating agent, a flavorant, and acolorant. Suitable pharmaceutical carriers are described in “Remington'sPharmaceutical Sciences” by E. W. Martin.

The abbreviations used in the nucleotide sequences throughout thisapplication are as follows: A=adenine, G=guanine, C=cytosine, T=thymine,U=uracil, R=purine (G or A), Y=pyrimidine (T or U or C), M=amino (A orC), S=strong interactions 3H-bonds (G or C), V=(A or C or G), K=(G orT), W=weak interactions 2H-bonds (A or T or U), N=any (A or G or C or Tor U), I=inosine.

Diagnostic Methods of the Invention

In one embodiment, the present invention provides a method fordiagnosing predisposition to obesity and associated conditions (e.g.,metabolic syndrome, diabetes mellitus, insulin-deficiency orinsulin-resistance related disorders, glucose intolerance, non-alcoholicfatty liver, abnormal lipid metabolism, and atherosclerosis) in a mammalby comparing the populations of Firmicutes and/or Eubacteria and/orBacteroidetes in the ileal microbiota of the mammal and in healthycontrols, wherein the increased populations of Firmicutes and/orEubacteria and/or Bacteroidetes in the ileal microbiota as compared tohealthy controls are indicative of predisposition to obesity andassociated conditions.

In another embodiment, the invention provides a method for diagnosingpredisposition to obesity and associated conditions (e.g., metabolicsyndrome, diabetes mellitus, insulin-deficiency or insulin-resistancerelated disorders, glucose intolerance, non-alcoholic fatty liver,abnormal lipid metabolism, and atherosclerosis) in a mammal by comparingthe levels populations of Firmicutes in the cecal and/or fecalmicrobiota of the mammal and in healthy controls, wherein the increasedlevel populations of Firmicutes in the cecal and/or fecal microbiota ascompared to healthy controls are indicative of predisposition to obesityand associated conditions.

In yet another embodiment, the invention provides a method fordiagnosing predisposition to obesity and associated conditions (e.g.,metabolic syndrome, diabetes mellitus, insulin-deficiency orinsulin-resistance related disorders, glucose intolerance, non-alcoholicfatty liver, abnormal lipid metabolism, and atherosclerosis) in a mammalby comparing the ratio of Firmicutes to Eubacteria (F/E ratio=relativeabundance of Firmicutes) in the cecal and/or fecal microbiota of themammal and in healthy controls, wherein the increased F/E ratio in thececal and/or fecal microbiota as compared to healthy controls isindicative of predisposition to obesity and associated conditions.

In a further embodiment, the invention provides a method for diagnosingpredisposition to obesity and associated conditions (e.g., metabolicsyndrome, diabetes mellitus, insulin-deficiency or insulin-resistancerelated disorders, glucose intolerance, non-alcoholic fatty liver,abnormal lipid metabolism, and atherosclerosis) in a mammal bydetermining the level of at least one of Coprobacillus, Sporacetigenium,Holdemania, Dorea, Blautia, Enterococcus, Erysipelotrichaceae IncertaeSedis (EIS), Clostridium cocleatum, and Peptosteptococcaceae IncertaeSedis (PIS) in the intestinal microbiota of the mammal, comparing thelevel to the level of the same bacteria in the intestinal microbiota ofhealthy controls, and identifying as a mammal predisposed to obesityetc. any mammal in which the level of at least one of said bacteria islower than in healthy controls.

In a separate embodiment, the invention provides a method for diagnosingpredisposition to obesity and associated conditions (e.g., metabolicsyndrome, diabetes mellitus, insulin-deficiency or insulin-resistancerelated disorders, glucose intolerance, non-alcoholic fatty liver,abnormal lipid metabolism, and atherosclerosis) in a mammal bydetermining the level of at least one of Johnsonella, Oscillibacter,Lachnospiraceae, Ruminococcaceae, and Clostridiales in the intestinalmicrobiota of the mammal, comparing the level to the level of the samebacteria in the intestinal microbiota of healthy controls, andidentifying as a mammal predisposed to obesity etc. any mammal in whichthe level of at least one of said bacteria is higher than in healthycontrols.

In another embodiment, the invention provides a method for diagnosingpredisposition to obesity and associated conditions (e.g., metabolicsyndrome, diabetes mellitus, insulin-deficiency or insulin-resistancerelated disorders, glucose intolerance, non-alcoholic fatty liver,abnormal lipid metabolism, and atherosclerosis) in a mammal by comparingthe total number of Butyryl CoA transferase (BCoAT)-encoding genes (orthe ratio of the total number of BCoAT-encoding genes to copies ofBacteroidetes 16S rRNA) in the intestinal microbiota of the mammal andin healthy controls, wherein the increased levels of BCoAT genes (or theincreased ratio of BCoAT-encoding genes to copies of Bacteroidetes 16SrRNA) as compared to healthy controls are indicative of predispositionto obesity and associated conditions.

Specific changes in microbiota can be detected using various methods,including without limitation quantitative PCR (qPCR) or high-throughputsequencing methods which detect over- and under-represented genes in thetotal bacterial population (e.g., 454-sequencing for communityanalysis), or transcriptomic or proteomic studies that identify lost orgained microbial transcripts or proteins within total bacterialpopulations. See, e.g., Eckburg et al., Science, 2005, 308:1635-8;Costello et al., Science, 2009, 326:1694-7; Grice et al., Science, 2009,324:1190-2; Li et al., Nature, 2010, 464: 59-65; Bjursell et al.,Journal of Biological Chemistry, 2006, 281:36269-36279; Mahowald et al.,PNAS, 2009, 14:5859-5864; Wikoff et al., PNAS, 2009, 10:3698-3703. Whileany number of suitable molecular techniques may be utilized,particularly useful molecular techniques for the purposes of the presentinvention include (i) screening of microbial 16S ribosomal RNAs (16SrRNA) using PCR and (ii) high-throughput “metagenome” sequencingmethods, which detect over- and under-represented genes in the totalbacterial population. Screening of 16S rRNA genes permits characterizingmicroorganisms present in the microbiota at the species, genus, family,order, class, or phylum level. Such screening can be performed, e.g., byconducting PCR using universal primers to the

V2, V3, V4, V6 (or V2-V4) region of the 16S rRNA gene followed byhigh-throughput sequencing and taxonomic analysis. See e.g., Gao et al.Proc. Natl. Acad. Sci. USA, 2007; 104:2927-32; Zoetendal et al., Mol.Microbiol., 2006, 59:1639-1650; Schloss and Handelsman, Microbiol. Mol.Biol. Rev., 2004, 68:686-691; Smit et al., Appl. Environ. Microbiol.,2001, 67:2284-2291; Harris and Hartley, J. Med. Microbiol., 2003,52:685-691; Saglani et al., Arch Dis Child, 2005, 90:70-73. Thehigh-throughput “metagenome” sequencing methods involve obtainingmultiple parallel short sequencing reads looking for under- andover-represented genes in a total mixed sample population. Suchsequencing is usually followed by determining the G+C content ortetranucleotide content (Pride et al., Genome Res., 2003, 13;145) of thegenes to characterize the specific bacterial species in the sample.Additional techniques include those involving cultivation of individualmicroorganisms from mixed samples. See, e.g., Manual of ClinicalMicrobiology, 8th edition; American Society of Microbiology, WashingtonDC, 2003.

Therapeutic Methods of the Invention

In conjunction with the diagnostic methods, the present invention alsoprovides therapeutic methods for treating obesity, metabolic syndrome,insulin-deficiency or insulin-resistance related disorders, glucoseintolerance, diabetes mellitus, non-alcoholic fatty liver, abnormallipid metabolism, atherosclerosis, and related disorders by restoringmammalian bacterial intestinal microbiota to the composition observed inhealthy subjects.

In certain specific embodiments, restoring of microbiota is achieved byadministering to a mammal in need thereof a therapeutically effectiveamount of a probiotic composition comprising an effective amount of atleast one bacterial strain, or a combinations of several strains, or aprebiotic composition, or a mixture thereof, wherein the composition (i)stimulates or inhibits specific metabolic pathways involved in hostenergy homeostasis and/or (ii) stimulates growth and/or activity ofbacteria which are under-represented in a disease and/or (iii) inhibitsgrowth and/or activity of bacteria which are over-represented in adisease.

In one embodiment, the present invention provides a method for promotingweight loss, preventing or treating obesity and associated conditions(e.g., metabolic syndrome, diabetes mellitus, insulin-deficiency orinsulin-resistance related disorders, glucose intolerance, non-alcoholicfatty liver, abnormal lipid metabolism, and atherosclerosis) in a mammalby administering a probiotic or a prebiotic composition or a combinationthereof, that stimulates growth or activity of at least one ofCoprobacillus, Sporacetigenium, Holdemania, Dorea, Blautia,Enterococcus, Erysipelotrichaceae Incertae Sedis (EIS), Clostridiumcocleatum, and Peptosteptococcaceae Incertae Sedis (PIS) in theintestinal microbiota of the mammal. In a related embodiment, theinvention provides a method for determining whether weight loss can beachieved or obesity and associated conditions can be treated in a mammalby the latter method by determining the level of at least one ofCoprobacillus, Sporacetigenium, Holdemania, Dorea, Blautia,Enterococcus, Erysipelotrichaceae Incertae Sedis (EIS), Clostridiumcocleatum, and Peptosteptococcaceae Incertae Sedis (PIS) in theintestinal microbiota of the mammal and comparing said level to thelevel of the same bacteria in the intestinal microbiota of healthycontrols, and identifying as a mammal treatable by the latter method anymammal in which the level of at least one of said bacteria is lower thanin healthy controls.

In another embodiment, the invention provides a method for promotingweight loss, preventing or treating obesity and associated conditions(e.g., metabolic syndrome, diabetes mellitus, insulin-deficiency orinsulin-resistance related disorders, glucose intolerance, non-alcoholicfatty liver, abnormal lipid metabolism, and atherosclerosis) in a mammalby inhibiting growth or activity of at least one of Johnsonella,Oscillibacter, Lachnospiraceae, Ruminococcaceae, and Clostridiales inthe intestinal microbiota of the mammal (e.g., by administering a narrowspectrum antibiotic, or another anti-bacterial agent, including aprobiotic [e.g., at least one of Coprobacillus, Sporacetigenium,Holdemania, Dorea, Blautia, Enterococcus, Erysipelotrichaceae IncertaeSedis (EIS), Clostridium cocleatum, and Peptosteptococcaceae IncertaeSedis (PIS)] which competes with at least one of Johnsonella,Oscillibacter, Lachnospiraceae, Ruminococcaceae, and Clostridiales formetabolic substrates, physical niches, or produces relevantantibiotic(s)). In a related embodiment, the invention provides a methodfor determining whether weight loss can be achieved or obesity andassociated conditions can be treated in a mammal by the latter method bydetermining the level of at least one of Johnsonella, Oscillibacter,Lachnospiraceae, Ruminococcaceae, and Clostridiales in the intestinalmicrobiota of the mammal and comparing said level to the level of thesame bacteria in the intestinal microbiota of a healthy control, andidentifying as a mammal treatable by the latter method any mammal inwhich the level of at least one of said bacteria is higher than inhealthy controls.

In yet another embodiment, the invention provides a method for promotingweight loss, preventing or treating obesity and associated conditions(e.g., metabolic syndrome, diabetes mellitus, insulin-deficiency orinsulin-resistance related disorders, glucose intolerance, non-alcoholicfatty liver, abnormal lipid metabolism, and atherosclerosis) in a mammalby administering a probiotic or a prebiotic composition or a combinationthereof, that lowers the populations of Firmicutes and/or Eubacteriaand/or Bacteroidetes in the ileal microbiota of the mammal.

In a further embodiment, the invention provides a method for promotingweight loss, preventing or treating obesity and associated conditions(e.g., metabolic syndrome, diabetes mellitus, insulin-deficiency orinsulin-resistance related disorders, glucose intolerance, non-alcoholicfatty liver, abnormal lipid metabolism, and atherosclerosis) in a mammalby administering a probiotic or a prebiotic composition or a combinationthereof, that lowers the populations of Firmicutes in the cecal and/orfecal microbiota of the mammal.

In a separate embodiment, the invention provides a method for promotingweight loss, preventing or treating obesity and associated conditions(e.g., metabolic syndrome, diabetes mellitus, insulin-deficiency orinsulin-resistance related disorders, glucose intolerance, non-alcoholicfatty liver, abnormal lipid metabolism, and atherosclerosis) in a mammalby administering a probiotic or a prebiotic composition or a combinationthereof, that lowers the ratio of Firmicutes to Eubacteria (F/Eratio=relative abundance of Firmicutes) in the cecal and/or fecalmicrobiota of the mammal.

In another embodiment, the invention provides a method for promotingweight loss, preventing or treating obesity and associated conditions(e.g., metabolic syndrome, diabetes mellitus, insulin-deficiency orinsulin-resistance related disorders, glucose intolerance, non-alcoholicfatty liver, abnormal lipid metabolism, and atherosclerosis) in a mammalby administering a probiotic or a prebiotic composition or a combinationthereof, that lowers the levels of Butyryl CoA transferase (BCoAT)enzyme in the intestinal microbiota of the mammal. In a relatedembodiment, the invention provides a method for promoting weight loss,preventing or treating obesity and associated conditions in a mammal byadministering a probiotic or a prebiotic composition or a combinationthereof, that lowers the ratio of the total number of Butyryl CoAtransferase (BCoAT)-encoding genes to copies of Bacteroidetes 16S rRNAin the intestinal microbiota of the mammal. In another relatedembodiment, the invention provides a method for promoting weight loss,preventing or treating obesity and associated conditions in a mammal byadministering a probiotic or a prebiotic composition or a combinationthereof, that lowers the levels of butyrate (e.g., measured usingchromatographic methods [see, e.g., Renom et al., Clin. Chem. Lab. Med.,2001, 39(1): 15-19]) in the intestinal microbiota of the mammal.

Probiotic and Prebiotic Compositions, Dosages and Administration

In conjunction with the above-identified therapeutic methods, thepresent invention provides probiotic and prebiotic compositions orcombinations of prebiotics and probiotics useful for promoting weightloss and/or treating obesity and associated conditions (e.g., metabolicsyndrome, diabetes mellitus, insulin-deficiency or insulin-resistancerelated disorders, glucose intolerance, non-alcoholic fatty liver,abnormal lipid metabolism, and atherosclerosis).

Probiotics useful in the methods of the present invention can compriselive bacterial strains and/or spores. In a preferred embodiment, suchlive bacterial strains and/or spores are from the genus Coprobacillus,Sporacetigenium, Holdemania, Erysipelotrichaceae Incertae Sedis (EIS),Clostridium cocleatum, or Peptosteptococcaceae Incertae Sedis (PIS). Incertain embodiments, the bacteria administered in the therapeuticmethods of the invention comprise one or more of Coprobacillus,Sporacetigenium, Holdemania, Erysipelotrichaceae Incertae Sedis (EIS),Clostridium cocleatum, and Peptosteptococcaceae Incertae Sedis (PIS) andone or more additional bacterial strains (such as, e.g., Oxalobacterspecies, Lactobacillus species, etc.).

One or several different bacterial inoculants can be administeredsimultaneously or sequentially (including administering at differenttimes). Such bacteria can be isolated from microbiota and grown inculture using known techniques. However, many bacterial species are verydifficult to culture and administration of others may lead to variousundesirable side-effects. The present invention therefore also comprisesadministering “bacterial analogues”, such as recombinant carrier strainsexpressing one or more heterologous genes derived from the bacteriaaffected in a disease. The use of such recombinant bacteria may allowthe use of lower therapeutic amounts due to higher protein expressionand may simultaneously minimize any potential harmful side-effectsassociated with reintroduction of specific bacterial strains.Non-limiting examples of recombinant carrier strains useful in themethods of the present invention include E. coli and Lactobacillus,Bacteroides and Oxalobacter. Methods describing the use of bacteria forheterologous protein delivery are described, e.g., in U.S. Pat. No.6,803,231.

In certain embodiments, a conditional lethal bacterial strain can beutilized as the inoculant or to deliver a recombinant construct. Such aconditional lethal bacteria survives for a limited time typically whenprovided certain nutritional supplements. It is contemplated that such asupplement could be a liquid, formulated to contain the nutritionalcomponent necessary to keep the bacteria alive. It is furthercontemplated that a patient/subject would drink such a supplement inintervals to keep the bacteria alive. Once the supplement is depleted,the conditional lethal bacteria dies. Methods relating to conditionallethal strains of H. pylori are described in U.S. Pat. No. 6,570,004.

In certain embodiments, the bacterial inoculant used in the methods ofthe invention further comprises a buffering agent. Examples of usefulbuffering agents include sodium bicarbonate, juice, milk, yogurt, infantformula, and other dairy products.

Administration of a bacterial inoculant can be accomplished by anymethod likely to introduce the organisms into the desired location. In apreferred embodiment, bacteria are administered orally. Alternatively,bacteria can be administered rectally, by enema, byesophagogastroduodenoscopy, colonoscopy, nasogastric tube, or orogastrictube.

The bacteria can be mixed with an excipient, diluent or carrier selectedwith regard to the intended route of administration and standardpharmaceutical practice. For easier delivery to the digestive tract,bacteria can be applied to liquid or solid food, or feed or to drinkingwater. For oral administration, bacteria can be also formulated in acapsule. The excipient, diluent and/or carrier must be “acceptable” inthe sense of being compatible with the other ingredients of theformulation and should be non-toxic to the bacteria and thesubject/patient. Preferably, the excipient, diluent and/or carriercontains an ingredient that promotes viability of the bacteria duringstorage. The formulation can include added ingredients to improvepalatability, improve shelf-life, impart nutritional benefits, and thelike. Acceptable excipients, diluents, and carriers for therapeutic useare well known in the pharmaceutical art, and are described, forexample, in Remington: The Science and Practice of Pharmacy. LippincottWilliams & Wilkins (A. R. Gennaro edit. 2005). The choice ofpharmaceutical excipient, diluent, and carrier can be selected withregard to the intended route of administration and standardpharmaceutical practice.

The dosage of the bacterial inoculant or compound of the invention willvary widely, depending upon the nature of the disease, the patient'smedical history, the frequency of administration, the manner ofadministration, the clearance of the agent from the host, and the like.The initial dose may be larger, followed by smaller maintenance doses.The dose may be administered as infrequently as weekly or biweekly, orfractionated into smaller doses and administered daily, semi-weekly,etc., to maintain an effective dosage level. It is contemplated that avariety of doses will be effective to achieve colonization of theintestinal tract with the desired bacterial inoculant, e.g. 10⁶, 10⁷,10⁸, 10⁹, and 10¹⁰CFU for example, can be administered in a single dose.Lower doses can also be effective, e.g., 10⁴, and 10⁵ CFU.

Non-limiting examples of prebiotics useful in the methods of the presentinvention include xylose, arabinose, ribose, galactose, rhamnose,cellobiose, fructose, lactose, salicin, sucrose, glucose, esculin, tween80, trehalose, maltose, mannose, mellibiose, raffinose,fructooligosaccharides (e.g., oligofructose, inulin, inulin-typefructans), galactooligosaccharides, amino acids, alcohols, water-solublecellulose derivatives (most preferably, methylcellulose, methyl ethylcellulose, hydroxyethyl cellulose, ethyl hydroxyethyl cellulose,cationic hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxyethylmethylcellulose, hydroxypropyl methylcellulose, and carboxymethylcellulose), water-insoluble cellulose derivatives (most preferably,ethyl cellulose), unprocessed oatmeal, metamucil, all-bran, and anycombinations thereof.

Table 1 provides a chart of prebiotics useful for stimulating growth andmetabolic activity (by acting as substrate for fermentation) ofCoprobacillus, Sporacetigenium, Holdemania, or Clostridium cocleatumbased on information from Kageyama et al., Microbiol. Immunol., 2000,44:23-28; Chen et al., Int J Syst Evol Microbiol, 2006, 56:721-725;Moore et al., Int J Syst Bact, 1997, 47(4):1201-1204, Willems et al.,Int J Syst Bact, 1995, 45:855-857; Lino et al., Int J Syst EvolMicrobiol, 2007, 57:1840-1845; Kaneuchi et al., Int J Syst Bact, 1979,29, 1. As follows from Table 1, trehalose, cellobiose, lactose, maltose,mannose, sucrose, fructose, galactose, salicin, mellibiose, andraffinose stimulate growth and metabolic activity of two or more generaselected from Coprobacillus, Sporacetigenium, Holdemania, or Clostridiumcocleatum, but not of Johnsonella and Oscillibacter.

TABLE 1 Coprobacillus Holdemania Sporacetegenium Clostridium cocleatumJohnsonella Oscillibacter Arabinose − w + − − +/−^(a) Cellobiose + w w +− − Esculin − + − Fructose + + + + − − Galactose + w + −Glucose + + + + + + Inulin + Lactose + + w + − − Maltose + + + + − −Mannose + w w + − − Mellibiose w w + − − Raffinose − w w + − − Rhamnose− w w − − − Ribose − w +/− − − + Salicin + + − w − − Sucrose + + w + − −Trehalose + w w − − − Xylose − w + − − + + facilitates growth, w weakgrowth promotion, +/− growth variable dependent on strain, − no growtheffect. Bold facilitates growth in >2 CSH organisms. ^(a)+ forL-arabinose; − for D-arabinose.

Preferred water-soluble cellulose derivatives for use in the presentinvention are water-soluble cellulose esters and cellulose ethers.Preferred cellulose ethers are water-soluble carboxy-C₁-C₃-alkylcelluloses, such as carboxymethyl celluloses; water-solublecarboxy-C₁-C₃-alkyl hydroxy-C₁-C₃-alkyl celluloses, such ascarboxymethyl hydroxyethyl celluloses; water-soluble C₁-C₃-alkylcelluloses, such as methylcelluloses; water-soluble C₁-C₃-alkylhydroxy-C₁₋₃-alkyl celluloses, such as hydroxyethyl methylcelluloses,hydroxypropyl methylcelluloses or ethyl hydroxyethyl celluloses;water-soluble hydroxy-C₁₋₃-alkyl celluloses, such as hydroxyethylcelluloses or hydroxypropyl celluloses; water-soluble mixedhydroxy-C₁-C₃-alkyl celluloses, such as hydroxyethyl hydroxypropylcelluloses, water-soluble mixed C₁-C₃-alkyl celluloses, such as methylethyl celluloses, or water-soluble alkoxy hydroxyethyl hydroxypropylcelluloses, the alkoxy group being straight-chain or branched andcontaining 2 to 8 carbon atoms. The more preferred cellulose ethers aremethylcellulose, methyl ethyl cellulose, hydroxyethyl cellulose, ethylhydroxyethyl cellulose, cationic hydroxyethyl cellulose, hydroxypropylcellulose, hydroxyethyl methylcellulose, hydroxypropyl methylcellulose,and carboxymethyl cellulose, which are classified as water-solublecellulose ethers by the skilled artisans. The most preferredwater-soluble cellulose ethers are methylcelluloses with a methyl molarsubstitution DS_(methoxyl) of from 0.5 to 3.0, preferably from 1 to 2.5,and hydroxypropyl methylcelluloses with a DS_(methoxyl) of from 0.9 to2.2, preferably from 1.1 to 2.0, and a MS_(hydroxypropoxyl) of from 0.02to 2.0, preferably from 0.1 to 1.2. The methoxyl content of methylcellulose can be determined according to ASTM method D 1347-72(reapproved 1995). The methoxyl and hydroxypropoxyl content ofhydroxypropyl methylcellulose can be determined by ASTM method D-2363-79(reapproved 1989). Methyl celluloses and hydroxypropyl methylcelluloses,such as K250M, K100M, K4M, K1M, F220M, F4M and J4M hydroxypropylmethylcellulose are commercially available from The Dow ChemicalCompany).

Preferred cationic hydroxyethyl celluloses are those described in U.S.Pat. No. 3,472,840. Preferably the cationic hydroxyethyl celluloses havegroups of the formula [R¹R²R³R⁴N⁺] (A_(z−))_(l/z), (II), wherein R¹, R²and R³ each independently are C₁₋₆-alkyl, preferably —CH₃ or —C₂H₅, R⁴is —CH₂—CHOH—CH₂— or —CH₂CH(OH)—, A^(z−)is an anion, and z is 1, 2 or 3.The cationic degree of substitution (often referred to as the CSorcationic substitution) of the cationic hydroxyethyl cellulose is in arange from about 0.075 to about 0.8, preferably about 0.15 to about0.60. A range of about 0.15 to about 0.60 corresponds to a Kjeldahlnitrogen content of about 0.8% to about 2.5%. More preferably, thecationic hydroxyethyl cellulose has a Kjeldahl nitrogen content between1.5 and 2.2%, which corresponds to a CS of about 0.3 to about 0.5. Inone embodiment, the cationic hydroxyethylcellulose has a Brookfield LVTdetermined solution viscosity of from about 5 cP (=mPa.s) to about10,000 cP, preferably from about 5 cP to about 3,000 cP, measured as aone weight percent aqueous solution at 25° C.

Combinations of two or more water-soluble cellulose derivatives are alsouseful. The water-soluble cellulose derivative generally has a viscosityof from 5 to 2,000,000 cps (=mPa.s), preferably from 50 cps to 1,000,000cps, more preferably from 1,000 to 300,000 cps, measured as a two weightpercent aqueous solution at 20° C. The viscosity can be measured in arotational viscometer.

Preferred water-insoluble cellulose derivatives for use in the presentinvention are water-insoluble cellulose ethers, particularly ethylcellulose, propyl cellulose or butyl cellulose. Other usefulwater-insoluble cellulose derivatives are cellulose derivatives whichhave been chemically, preferably hydrophobically, modified to providewater insolubility. Chemical modification can be achieved withhydrophobic long chain branched or non-branched alkyl, arylalkyl oralkylaryl groups. “Long chain” typically means at least 5, moretypically at least 10, particularly at least 12 carbon atoms. Otherstype of water-insoluble cellulose are crosslinked cellulose, whenvarious crosslinking agents are used. Chemically modified, including thehydrophobically modified, water-insoluble cellulose derivatives areknown in the art. They are useful provided that they have a solubilityin water of less than 2 grams, preferably less than 1 gram, in 100 gramsof distilled water at 25° C. and 1 atmosphere. The most preferredcellulose derivative is ethyl cellulose. The ethyl cellulose preferablyhas an ethoxyl substitution of from 40 to 55 percent, more preferablyfrom 43 to 53 percent, most preferably from 44 to 51 percent. Thepercent ethoxyl substitution is based on the weight of the substitutedproduct and determined according to a Zeisel gas chromatographictechnique as described in ASTM D4794-94(2003). The molecular weight ofthe ethyl cellulose is expressed as the viscosity of a 5 weight percentsolution of the ethyl cellulose measured at 25° C. in a mixture of 80volume percent toluene and 20 volume percent ethanol. The ethylcellulose concentration is based on the total weight of toluene, ethanoland ethyl cellulose. The viscosity is measured using Ubbelohde tubes asoutlined in ASTM D914-00 and as further described in ASTM D446-04, whichis referenced in ASTM D914-00. The ethyl cellulose generally has aviscosity of up to 400 mPa's, preferably up to 300 mPa's, morepreferably up to 100 mPa's, measured as a 5 weight percent solution at25° C. in a mixture of 80 volume percent toluene and 20 volume percentethanol. The preferred ethyl celluloses are premium grades ETHOCEL ethylcellulose which are commercially available from The Dow Chemical Companyof Midland, Mich. Combinations of two or more water-insoluble cellulosederivatives are also useful. Preferably the water-insoluble cellulosederivative has an average particle size of less than 0.1 millimeter,more preferably less than 0.05 millimeter, most preferably less than0.02 millimeter. Preferably the water-insoluble cellulose derivative isexposed to an edible fat or oil before being administered to anindividual so that the cellulose derivative imbibes the fat or oil.Advantageously the water-insoluble cellulose derivative is exposed to anexcess of the fat or oil at about 40 to 60° C.

In certain other specific embodiments, the therapeutic methods of theinvention rely on the administration of a therapeutically effectiveamount of a naturally or recombinantly produced bacterial protein or acombination of such proteins which (i) increase the number and/oractivity of one or more bacteria which are under-represented in adisease and/or (ii) decrease the number and/or activity of one or morebacteria which are over-represented in a disease. The proteins accordingto this embodiment may be produced by the same strain of bacteria whichis intended to be regulated or by a different strain.

Prior to administering to humans, the effectiveness of the noveltherapeutic compositions of the present invention can be studied inanimal models of obesity, such as, e.g., sub-therapeutic antibiotictreatment (STAT) mice (Cho et al., Gastroenterology, 2009, 136(5)Supplement 1: A-102), ob/ob mice (Ley et al., Proc. Natl. Acad. Sci. USA2005; 102:11070-5; Turnbaugh et al., Nature 2006; 444:1027-31), db/dbmice (Kobayashi et al., Metabolism, 2000, 48(1):22-31), diet-inducedobesity (DIO) mice (Petro et al., Metabolism, 2004, 53(4):454-457), NODmice (Wen et al., Nature, 2008; 455(7216):1109-1113), etc.

Combination Treatments

For an enhanced therapeutic effect, the probiotics and/or prebiotics asdescribed herein can be administered in combination with othertherapeutic agents or regimes as discussed. The choice of therapeuticagents that can be co-administered with the probiotics and/or prebioticsof the invention depends, in part, on the condition being treated.

Non-limiting examples of additional pharmaceutically active compoundsuseful for treatment of obesity, metabolic syndrome, insulin-deficiencyor insulin-resistance related disorders, glucose intolerance, diabetesmellitus, non-alcoholic fatty liver, abnormal lipid metabolism,atherosclerosis, and related disorders include anti-inflammatory agents,antioxidants, antiarrhythmics, cytokines, analgesics, vasodilators,antihypertensive agents including beta-blockers, angiotensin convertingenzyme inhibitors (ACE inhibitors), and calcium channel blockers,inhibitors of cholesterol synthesis, cholesterol binding agents,antithrombotic agents, central modulators of appetite, and diabetesdrugs. Examples of inhibitors of cholesterol synthesis or absorptionwhich are useful in the combination therapies of the present inventioninclude Hmg-CoA reductase inhibitors and their bio-active metabolites,such as, e.g., simvastatin, lovastatin, pravastatin, compactin,fluvastatin, dalvastatin, atorvastatin, HR-780, GR-95030, CI-981, BMY22089, and BMY 22566. See, e.g., U.S. Pat. Nos. 4,346,227; 4,444,784;4,857,522; 5,190,970; 5,316,765, and 5,461,039; PCT Publ. No.W084/02131; GB Pat. No. 2,202,846. As used in the methods orcompositions of the present invention, any one or several of the Hmg-CoAreductase inhibitor compounds may be mixed with L-arginine or asubstrate precursor to endogenous nitric oxide, as described in U.S.Pat. Nos. 6,425,881 and 6,239,172, and 5,968,983, to provide atherapeutically effective mixture for use in conjunction with probioticsand/or prebiotics of the present invention.

Non-limiting examples of diabetes drugs useful in the combinationtherapies of the present invention include insulin, proinsulin, insulinanalogs, activin, glucagon, somatostatin, amylin, actos (pioglitazone),amaryl (glimepiride), glipizide, avandia (rosiglitazone), glucophage,glucotrol, glucovance (a combination of glyburide and metformin), andthe like. See, e.g., U.S. Pat. No. 6,610,272. The term “insulin”encompasses natural extracted human insulin, recombinantly producedhuman insulin, insulin extracted from bovine and/or porcine sources,recombinantly produced porcine and bovine insulin and mixtures of any ofthese insulin products. In accordance with the present invention,administering probiotics and/or prebiotics of the present invention incombination with insulin is expected to lower the dose of insulinrequired to manage the diabetic patient, while also alleviating thesymptoms of metabolic syndrome.

In accordance with the present invention there may be numerous tools andtechniques within the skill of the art, such as those commonly used inmolecular immunology, cellular immunology, pharmacology, andmicrobiology. Such tools and techniques are describe in detail in e.g.,Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual. 3rd ed.Cold Spring Harbor Laboratory Press: Cold Spring Harbor, N.Y.; Ausubelet al. eds. (2005) Current Protocols in Molecular Biology. John Wileyand Sons, Inc.: Hoboken, N.J.; Bonifacino et al. eds. (2005) CurrentProtocols in Cell Biology. John Wiley and Sons, Inc.: Hoboken, N.J.;Coligan et al. eds. (2005) Current Protocols in Immunology, John Wileyand Sons, Inc.: Hoboken, N.J.; Coico et al. eds. (2005) CurrentProtocols in Microbiology, John Wiley and Sons, Inc.: Hoboken, N.J.;Coligan et al. eds. (2005) Current Protocols in Protein Science, JohnWiley and Sons, Inc. Hoboken, N.J.; and Enna et al. eds. (2005) CurrentProtocols in Pharmacology, John Wiley and Sons, Inc.: Hoboken, N.J.

EXAMPLES

The present invention is also described and demonstrated by way of thefollowing examples. However, the use of these and other examplesanywhere in the specification is illustrative only and in no way limitsthe scope and meaning of the invention or of any exemplified term.Likewise, the invention is not limited to any particular preferredembodiments described here. Indeed, many modifications and variations ofthe invention may be apparent to those skilled in the art upon readingthis specification, and such variations can be made without departingfrom the invention in spirit or in scope. The invention is therefore tobe limited only by the terms of the appended claims along with the fullscope of equivalents to which those claims are entitled.

Example 1 Analysis of Diet-Associated Changes in Intestinal Microbiotaof Mice Materials and Methods 1. Animals and Diets

Thirty (30) obese male C57/B16J mice (from Jackson Laboratories, BarHarbor, Me.) were studied. All mice were fed a high fat (60% fat) diet(also termed diet-induced obesity [DIO] diet; supplied by Research DietsInc., New Brunswick, N.J.) and water ad libitum for at least two months.Then baseline fecal samples were obtained and animals were divided inthree groups of ten (10) mice each. One group was maintained on thehigh-fat (60% fat) diet, one group was converted to a low fat (10% fat)diet (also supplied by Research Diets Inc., New Brunswick, N.J.), andthe third group was fed a 60% fat diet+HPMC. Hydroxypropylmethylcellulose (HPMC) was present at 8 percent weight level in thetreatment diet. It was mixed with the powdered components of the diet.The HPMC had a methoxyl content of 19-24 percent, a hydroxypropoxylcontent of 7-12 percent and a viscosity of about 250,000 mPa's, measuredas a 2 wt. % aqueous solution at 20° C., and is commercially availablefrom The Dow Chemical Company under the Trademark METHOCEL K250Mhypromellose. Animals were weighted periodically. A fresh fecal pelletwas collected from each individual mouse at baseline, after 2 and 4weeks, and shortly after sacrifice and frozen at −80° C. At the time ofsacrifice, the cecal and ileal contents were also frozen and stored at−80° C. for future study.

2. DNA extraction

Approximately 10 mg each of the fecal, cecal, and ileal samples wereextracted using the MoBio Powersoil 2 DNA Isolation kit (MoBioLaboratories, Carlsbad, Calif. ) as per the manufacturer's instructions.This extraction method uses a combination of mechanical disruption usingbead-beating and spin filtration using silica filter tubes to extractbacterial genomic DNA from each of the samples.

3. qPCR, sequencing and taxonomic analysis

DNA extracted from cecal, ileal, and fecal specimens was subjected toPCR using barcoded universal primers interrogating regions V3-V5 of the16S rRNA gene followed by 454 sequencing and taxonomic analysis.

Total eubacterial levels were determined by a standardized quantitativePCR (qPCR) using primers Eub519F: 5′-CAGCAGCCGCGGTRATA-3′ (SEQ ID NO: 1)and Eu785R: 5′-GGACTACCVGGGTATCTAAKCC-3′ (SEQ ID NO: 2) directed toconserved 16S rRNA fragments.

PCR reaction mixture and program (Power SYBR Green) were as follows:

Reagents Stock Vol/Reac (μl) Final PCR Master 2x 12.5 1x Mix F. primer10 μM 1 0.4 μM R. primer 10 μM 1 0.4 μM BSA 20 ng/μl 0.125 0.1 ng/μlTemplate 1 Total 25

-   Program: 50° C. 2 min, 95° C. 10 min

95° C. 15 Seconds and

56° C. 60 Seconds 40 cycles

Firmicutes levels were determined by qPCR using primers Firm934F:5′-GGAGYATGTGGTTTAATTCGAAGCA-3′ (SEQ ID NO: 3) and Firm1060R:5′-AGCTGACGACAACCATGCAC-3′ (SEQ ID NO: 4) directed to conserved 16S rRNAfragments.

PCR reaction and Program (Power SYBR Green) were as follows:

Reagents Stock Vol/Reac (μl) Final PCR Master 2x 12.5 1x Mix F. primer10 μM 1 0.4 μM R. primer 10 μM 1 0.4 μM BSA 20 ng/μl 0.125 0.1 ng/μlTemplate 1 Total 25

-   Program: 50° C. 2 min, 95° C. 10 min

95° C. 15 Seconds and

60° C. 60 Seconds 40 cycles

Bacteroidetes levels were determined by qPCR using primers Bact934F:5′-GGARCATGTGGTTTAATTCGATGAT -3′ (SEQ ID NO: 5) and Bact1060R:5′-AGCTGACGACAACCATGCAG -3′ (SEQ ID NO: 6) directed to conserved 16SrRNA fragments.

PCR reaction and Program (Power SYBR Green) were as follows:

Reagents Stock Vol/Reac (μl) Final PCR Master 2x 12.5 1x Mix F. primer10 μM 1 0.4 μM R. primer 10 μM 1 0.4 μM BSA 20 ng/μl 0.125 0.1 ng/μlTemplate 1 Total 25

-   Program: 50° C. 2 min, 95° C. 10 min

95° C. 15 Seconds and

60° C. 60 seconds 40 cycles

The samples then underwent 454 pyrosequencing (Roche) using barcodedprimers designed to interrogate the 16S rRNA regions V3-V5. The averagenumber of sequence reads obtained from fecal pellets were 5671±1981reads, while the average number of sequence reads obtained from cecaland ileal samples were 4901±2271 and 6662±2438, respectively. The totalamount of data generated in the sequencing experiment was about 0.18 Gb(180 Mb). Sequence data were summated to the phylum, class, order,family and genus levels and analyzed.

Total fungal levels were determined by qPCR using primers directed tothe conserved ITS2 region in the fungal rrn operon ITS1FCTYGGTCATTTAGAGGAAGTAA (SEQ ID NO: 7) and ITS2 RCTGCGTTCTTCATCGWTG (SEQID NO: 8) and probe TCYGTAGGTGAACCTGCRG (SEQ ID NO: 9).

The total number of genes encoding Butyryl CoA transferase (BCoAT),regardless of the taxonomic origin of the gene, were determined by qPCRusing primers BCoATscrF GCIGAICATTTCACITGGAAYWSITGGCAYATG (SEQ ID NO:10) and BCoATscrR CCTGCCTTTGCAATRTCIACRAANGC (SEQ ID NO: 11). BCoAT genenumber was determined by quantitative PCR using FastStart SYBR GreenMaster Mix (Roche) with primer concentration at 500 nM. Samples were runat the following temperature profile: 50° C. for 2 minutes, 95° C. for10 minutes, then cycle 40 times at 95° C. for 15 seconds, 53° C. for 30seconds, and 72° C. for 30 seconds. Positive samples were confirmed bymelting curve analysis.

Bioinformatic Pipeline 1. After the completion of sequencing, a readprocessing pipeline consisting of a set of modular scripts designed atthe JCVI were employed for deconvolution, trimming, and qualityfiltering. First reads were deconvoluted or assigned to samples based ontheir unique 10 nt barcode allowing no more than a one nt mismatch tothe barcode. After deconvolution, barcode and 16S primer sequences wereremoved allowing a maximum of 6 mismatches to the 16S primer and amaximum primer to barcode distance of 3 nt. Reads with an average lengthof <100 nt, and reads with ‘Ns’ were removed. A Blastn quality check wasperformed against an internal data set of 16S reads to remove any samplereads not consistent with 16S gene sequences in which at least 30% ofthe query must be covered by the alignment (60 nt minimum). Passingreads were subsequently checked for chimeras using a modified version ofthe RDP Chimera Check, using a reference data set maintained in-house.Remaining reads were then classified to lowest taxonomic level possibleusing the RDP Classifier with 80% confidence. Taxonomic results werethen converted to relative abundance or ratios for each sample, and thedifference between was calculated with the Mann-Whitney U test (means ±and ratios depicted in FIGS. 4-6).

Results Correlations Between Diets and Weight Gain

In the group which was continuously fed high fat (60% fat) diet, animalsgained weight. In the group where HPMC was added to the high fat (60%fat) diet, animals stopped gaining weight as soon as HPMC was added. Andanimals switched to the low fat (10% fat) diet lost weight. Theseobservations were consistent with prior observations of the role of HPMCin controlling metabolic syndrome, diabetes mellitus and obesity, and inpromotion of weight loss or maintenance of the desired body weight (seethe Background section, above).

Intestinal Populations of Microorganisms

The present inventors set to investigate whether weight gain associatedwith high fat (60% fat) diet and the absence of such weight gain uponthe addition of HPMC to the same diet correlates with changes inintestinal microbiota.

Eubacteria

As demonstrated in FIG. 3A, at baseline, mice had 9-10 log16S copies/gof fecal pellet without significant differences between the threegroups. At 2 weeks, Eubacterial (total bacteria) counts rose slightly inthe 10% fat and 60% fat groups, and fell in the 60% fat+HPMC group, butnone of the changes were statistically significant. By 4 weeks, levelsin the 10% fat and 60% fat groups were unchanged, but the 60% fat+HPMCgroup was significantly lower. The levels in the cecum at sacrifice werevery similar to the 4 week results, as expected, with the same lowertrends for the 60% fat+HPMC group. The ileal samples at sacrifice werelower, especially in the 60% fat+HPMC group. Thus, there is consistencyin the decrease observed in the 60% fat+HPMC group with respect to thebaseline, in the 2-week, 4-week, cecal, and ileal samples. These dataprovide evidence that adding HPMC to the diet lowers total Eubacterialpopulations with reference to the other two groups.

The Eubacterial populations within each group of 10 mice were alsoanalyzed over the study period. For the mice maintained on the 60% fatdiet, and the mice converted to the low fat (10% fat) diet, there wereno significant differences over time (comparing Basline, 2-week, and4-week samples). However, for the mice converted from the 60% fat dietto the 60% fat+HPMC diet, there was a progressive and significantdecline between Basline and 4-week samples. In all 3 diet groups, ileallevels were 0.5-1.0 log₁₀ lower than in cecum.

Firmicutes

As demonstrated in FIG. 3C, results for Firmicutes were generallysimilar to those for all Eubacteria. This internal consistency is notsurprising since Firmicutes represent the major population withinEubacteria in the mammalian intestinal tract. At baseline, theFirmicutes populations of all three groups were similar, as expected,but by the second week, the population in the 60% fat+HPMC group wastrending lower, and, in the 4-week sample, cecal and ileal samples weresignificantly lower than in the two other diet groups.

There were no differences over time in the group of mice maintained onthe 60% fat diet, or changed to the 10% fat diet, but there was aprogressive and significant decrease in the 60% fat+HPMC group. Ilealpopulations also were significantly lower (0.5-1.0) than in the cecalsamples.

Bacteroidetes

As demonstrated in FIG. 3B, Bacteroidetes populations were notsubstantially different in mice on the 3 diets at baseline or at 2weeks, however, by 4 weeks, and in the ileal samples, levels were lowerin the 60% fat+HPMC group than in the other two groups. After mice wereswitched from the 60% fat diet to the 10% fat diet, Bacteroidetes levelsrose significantly. Thus, changing from the 60% fat diet to a low fat(10%) diet or adding HPMC to the 60% fat diet perturbed theBacteroidetes numbers, but in apparently opposite directions.

Fungal populations

Fungal concentrations were much lower than were measures for Eubacteria,with a median of 4.0-5.0 log₁₀ 16S copies/g. There were no significantdifferences between the groups fed different diets or over time. Ilealconcentrations were higher than cecal, opposite to the Eubacterialconcentrations, but the differences were not significant.

Ratios Between Populations

Firmicutes/Eubacteria (F/E)

Firmicutes represented a median of 40% to 60% of the total bacterialpopulation in the fecal specimens. As shown in FIG. 3G, no trends overtime in the Firmicutes/Eubacteria (F/E) ratios were present comparingthe three groups of mice put on different diets. However, the mice whoswitched to the low (10%) fat diet had ratios that were significantlylower in the cecal samples than mice fed the 60% fat+HPMC diet, andhigher in the ileal samples than mice fed the 60% fat diet. Theintragroup comparisons did not show any significant differences for thefecal specimens over time.

Bacteroidetes/Eubacteria (B/E)

Inversely from the Firmicutes/Eubacteria (F/E) ratio, theBacteroidetes/Eubacteria (B/E) ratios in the cecal specimens weresignificantly higher for animals fed the 60% fat+HPMC diet than for micefed either the 10% fat or 60% fat diet (FIG. 3F). Changing the diet from60% fat at baseline to 10% fat also was accompanied by a significantincrease in the B/E ratio over 4 weeks, with the major increaseoccurring by 2 weeks.

BCoAT Studies

Microbes can contribute to obesity through fermentation ofnon-digestible carbohydrates in the colon to short chain fatty acids,such as acetate, butyrate, and propionate. See Bergman, Physiol Rev1990; 70:567-90; Wong et al., J Clin Gastroenterol 2006; 40:235-43;Pryde et al., FEMS Microbiol Lett 2002; 217:133-9; Wolfe, Microbiol MolBiol Rev 2005; 69:12-50. This process represents a 75% energy conversionto a product that is readily absorbed in the intestine, contributing 10%of host caloric intake. Butyrate is the preferred energy source forcolonocytes. Butyryl CoA transferase (BCoAT) is critical for butyratesynthesis. See, e.g., Charrier et al., Microbiol., 2006,152:179-85;Duncan et al., Appl. Environ. Microbiol., 2002, 68:5186-90; Louis andFlint, Appl. Environ. Microbiol., 2007, 73:2009-12. The BCoAT-encodinggene is widely conserved in intestinal bacteria.

The present inventors have hypothesized that the change in diet, and,specifically, the addition of HPMC, affects intestinal energymetabolism. This hypothesis was tested by examining the number of copiesof BCoAT genes, as well as the ratio of BCoAT genes, relative to majortaxonomic groups (FIGS. 3D and 3H).

Inter-Group Comparisons of BCoAT Copy Number

As shown in FIGS. 3D and 3H, at baseline, most fecal samples had between7 and 8 log₁₀ BCoAT copies detected, and, as expected, there were nosignificant differences between the three diet groups. After 2 weeks,the number of BCoAT copies in feces was significantly lower in the 60%fat+HPMC group versus the 60% fat group. After 4 weeks, the BCoATnumbers in feces were significantly lower than in both of the othergroups, which also was found in the cecal samples at sacrifice. Thus, in3 different groups of specimens, BCoAT populations were significantlydifferent after HPMC was added to the diet. In the ileal samples, thehighest BCoAT levels were in the 60% fat group, with significantly lowerlevels in the 10% fat and 60% fat+HPMC groups. Thus, these studiesconfirm the hypothesis that addition of HPMC lowers BCoAT levels inrelation to the other groups, in a manner predicted to lower butyrateavailability and energy production.

Intra-Group Comparisons of BCoAT Copy Number

There were no significant differences from baseline over 4 weeks in the10% fat and 60% fat groups. However, in the 60% fat+HPMC group, therewas a progressive and significant decline in BCoAT levels of about 1log₁₀ (90% reduction). This is both statistically and biologicallysignificant.

Relationship of BCoAT Copy Number to Taxonomic Findings-Inter-GroupAnalyses

There were no significant differences in the ratio of BCoAT genes tonumbers of total bacteria, Firmicutes, or Bacteroidetes, with threeexceptions. In the ileal samples, the BCoAT/total bacteria ratios andthe BCoAT/Firmicutes ratios were significantly higher in the 60%fat+HPMC group compared with the 10% fat group. In both cases, the 60%fat group was intermediate, but the differences were not significant.The BCoAT/Bacteroidetes ratios rose significantly from the 10% fat groupto the 60% fat+HPMC group. These results provide evidence that theenergy metabolism in proportion to the numbers of Firmicutes andBacteroidetes changed in the ileum depending on diet. No other changeswere significant.

Relationship of BCoAT Copy Number to Taxonomic Findings-Intra-GroupAnalyses

There were no significant differences between the baseline, 2 week, or 4week samples with only a single exception. Mice fed the 10% fat diet hada progressive and significant decrease in the BCoAT/Bacteroidetes ratiosover four weeks.

Comparison of Sequence Data at Baseline, 2 Weeks, and 4 Weeks

Sequence data extracted from fecal pellets obtained from study mice atbaseline, 2 weeks, and 4 weeks was compared using heat map (FIG. 18) andprincipal component analysis (PCA) plots (FIG. 16). At baseline, thedistribution of the mice based on the genus level microbial compositionof their fecal pellets was random. This was corroborated by unsupervisedhierarchical clustering analysis at the same taxonomic level in an NMDSanalysis. This was an expected result because all mice to this point hadbeen raised and fed under identical conditions (60% fat diet). After 2weeks of intervention, mice in each of the 3 study groups began tocluster, although the clustering was not statistically significant atthis intermediate time point. By 4 weeks, there was significantclustering of the mice into their respective study groups. In a heat mapanalysis, there were three distinct deep branch points (termed I, II,and III). In branch I, 7 of the 10 mice were from the 60% fat+HPMCgroup. In branch II, 9 of the 10 mice were from the 60% fat group. Inbranch III, 7 of the 10 mice were from the 10% fat group. The clusteringwas corroborated in NMDS plots, in which all the 60% fat+HPMC mice wereadjacent, and most of the 60% fat mice also were contiguous. These datademonstrate that there are significant and consistent effects of thediets on the intestinal microbiota of mice that are observable within 4weeks of initiation.

Comparison of Cecal and Ileal Samples

Comparison of the sequencing data obtained from ileal and cecal samplesalso was accomplished by heat map analysis. Ileal samples generated 2deep branch points containing 18 and 10 mice. In the larger branch of 18mice, 14 were exposed to the 60% fat diet (9 on 60% fat and 5 mice on60% fat+HPMC) while the branch of 10 mice was primarily composed of miceexposed to the 10% fat diet. The data obtained from the cecal samplesgenerated three branch points (termed I, II, and III). The most notablefinding is in branch III, in which 10 of 10 mice were exposed to the 60%fat+HPMC diet. Branch I consisted primarily of the 10% fat group (6/10mice) and branch II consisted of the 60% fat group (5/9 mice). Thesefindings suggest that HPMC exposure has a more significant effect on themicrobiome found in the cecum than in the ileum. This is consistent withthe fact that the microbial numbers in the cecum and thus theirmetabolic contributions are likely greater than anywhere else in thegastrointestinal tract (see, e.g., Turnbaugh et al., Nature 2006,444(7122):1027; Qu et al., PLoS One 2008, 3(38):e2945).

Alterations of the Taxonomic Composition Caused by Exposure to HPMC

Comparison of microbial abundance in the cecal samples obtained from 10%fat, 60% fat, and 60% fat+HPMC groups is shown in FIG. 4A and issummarized in FIG. 4B. In comparing the three study groups, thedifferences between the 10% fat and 60% fat groups were relativelymodest, with the most notable changes at the genus level, an increase inDorea, and decrease in Coprobacillus and Papillibacter was observed. Nochanges were found at higher taxonomic levels. However, when comparingthe 60% fat+HPMC group to the other two diets, there were marked andsignificant differences at multiple taxonomic levels. Notable changes ingenera are seen in the decrease of Oscillibacter (lino et al., Int. J.Syst. Evol. Microbiol., 2007, 57:1840-1845; Walker at al., ISME J.,2010, 1-11) and Johnsonella (Moore and Moore, Int. J. Syst. Bacteriol.,1994, 44(2):187-192) as well as in the marked increase in Coprobacillus(Kageyama and Benno, Microbiol. Immunol., 2000, 44(1):23-28),Sporacetigenium (Chen et al., Int. J. Systematic Evol. Microbiol., 2006,56:721-725), and Holdemania (Willems et al., Int. J. SystematicBacteriol., 1997, 47(4):1201-1204) (FIG. 5) and slight increase inDorea, Blautia, and Enterococcus.

By 454-pyrosequencing, at the family level, 60% fat+HPMC groups showed amarked increase in Erysipelotrichaeceae and Peptostreptococcaceae, aslight increase in Clostridiales Insertae Sedis XIV, andEnterococcaceae, and a decrease in Lachnospiraceae and Ruminococcaceaewhen compared to the 60% fat control mice. At the order level, there wasan increase in Erysipelotrichales and Lactobacillales, and a decrease inClostridiales. At the Class level, there was an increase inErysipelotriche and Bacilli, and a decrease in Clostridia. There was adownward trend in Firmicutes relative abundance (P=0.065 by Mann-WhitneyU) and an upward trend in Bacteroidetes relative abundance (P=0.076).There were no statistically significant changes between 60% fat+HPMC andthe 60% treatment groups at the phylum level, although the decrease inFirmicutes neared significance (p=0.065).

When cecal microbiota abundance in mice on 60% fat+HPMC diet is comparedto mice on the 10% fat diet, the changes in taxa are similar to thecomparison between 60% fat+HPMC and 60% fat with the followingexceptions. There is a significant increase in Bacteroidetes (phylum),Bacteroidia (class), Bacteroidales (order), and Anaerotruncus (genus) inthe 60%+HPMC group. There is no significant difference in ClostridialesInsertae Sedis XIV, Enterococcaceae (family), Blautia, or Enterococcus(genus).

Summary of 454 Sequencing Data

These data provide strong evidence that adding HPMC to a 60% dietchanges the population composition of the intestinal microbiota.Clustering of the groups based on sequence data demonstrates that thedietary interventions, both of 60% fat diet and 60% fat+HPMC diet,caused consistent and significant changes at the genus level in thecomposition of the gut microbiome. The data also show that the primaryeffect of the HPMC may be in the cecum, rather than in the ileum.Finally, the addition of HPMC to the diet caused significant shifts inthe composition of the gut microbiome, more than simply diet alone.

Conclusions

The above data provide evidence that adding HPMC to a 60% fat dietchanges the population size and composition of the intestinalmicrobiota. The data are internally consistent and show reductions intotal bacterial populations, primarily reflecting reduction inFirmicutes. Diet change also affects Bacteroidetes populations. The factthat the changes were in the same direction and to a similar degreesupports the hypothesis that adding HPMC affects the microbiota in waysequivalent to lowering dietary fat content. The 454 sequencing dataconfirmed the consistency of the findings.

The data provided herein with respect to specific changes also allows todevelop various diagnostic methods for predicting predisposition toweight gain on a high fat diet and effectiveness of fiber (e.g., HPMC)treatment for weight loss or weight gain prevention. As shown in FIGS.6A-6B, a useful diagnostic ratio at the genus level can be developed bydividing the sum of any combination of Coprobacillus, Sporacetigenium,and/or Holdemania (i.e., C+S+H or C+H or C+S or S+H or C or S or H) byany combination of Johnsonella and/or Oscillibacter (i.e., J+O or J orO), wherein a ratio below 1 indicates a state that is predisposed toweight gain while a ratio above 3 indicates a state that has a highpropensity to prevent weight gain. Additional diagnostic ratios can bedeveloped by dividing the sum of any combination of Coprobacillus,Sporacetigenium, and/or Holdemania (i.e., C+S+H or C+H or C+S or S+H orC or S or H) by the phylum Firmicutes (F), wherein a ratio below 0.1indicates a state that is predisposed to weight gain while a ratio above0.1 indicates a state that has a high propensity to prevent weight gain.As shown in FIG. 6C, useful diagnostic ratios can be also developed bydividing the sum of any combination of Erysipelotrichaceae and/orPeptostreptococcacea by Lachnospiraceae and/or Ruminococcaceae, whereina ratio below 0.1 indicates a state that is predisposed to weight gainwhile a ratio above 0.1 indicates a state that has a high propensity toprevent weight gain.

Example 2 A Cholesterol-Lowering Dietary Fiber Perturbs the MurineIntestinal Microbiota Materials and Methods

Animals and Diets were the same as in Example 1, supra.

Hepatic lipid analysis. Lyophilized liver samples were extracted usingan accelerated solvent extractor (Dionex ASE, Sunnyvale, Calif.) at 100°C., ˜13.8 MPa with 75/25 hexane/2-propanol, dried and weighed todetermine the percentage of total hepatic lipids, and hepatic totalcholesterol, free cholesterol, and triglyceride levels (RocheDiagnostic/Hitachi 914 clinical analyzer).

Fecal lipid analysis. Fecal lipids were extracted on a Dionex ASE systemusing a mixture of hexane and 2-propanol (3:2, v/v, 2% acetic acid) at15 MPa and 60° C. for 30 min, then divided into two aliquots. The firstsample was analyzed for saturated and unsaturated fatty acid compositionby GC separation. Briefly, the fatty acids in the lipid extract weremethylated using boron trifluoride methanol127, and the derivatizedsamples analyzed by gas chromatography Agilent 6890 series GC, with aflame ionization detector and a DB-23 analytical column (Agilent, SantaClara, Calif.). The initial oven temperature was 200° C. for 5 min, thenincreased to 250° C. at 5° C./min and held for 5 min. A calibrationsolution was prepared to contain 2 mg/mL of methyl palmitate (C16:0),methyl stearate (C18:0), methyloleate (C18:1), methyl linoleate (C18:2),and methyl linolenate (C18:3), and 11 μg/mL methyl erucate (Nu-chek,Elysian, Minn.) in heptane. The second aliquot was analyzed for totalbile acids and sterols using a modified chromatographic method28.Briefly, using a 1200RR HPLC system (Agilent) with an Acquity BEH C18column [1.7 μm, 2.1×100 mm; (Waters)], a reversed-phase separation wasperformed with a gradient of two mobile solvent phases: (A)methanol/acetonitrile/water (53:23:24, v/v/v) and (B) 2-propanol (100%).Crystalline ammonium acetate was added to each phase to form a 30 mMsolution. Solvent A was acidified by adding 2.4% (v/v) glacial aceticacid. A linear gradient at a flow rate of 0.25 mL/min was performed asfollows: 0-6 min, 4-36% B; 6-8 min, 36-48% B; 8-17 min, 48-51% B; 17-18min, 51-73% B; 18-31 min, 73-85% B; and 31-34 min, 85-96% B. In allexperiments, the columns were re-equilibrated between injections withthe initial mobile phase (10 mL). The LC effluent was monitored using aCorona Plus charged aerosol detection apparatus (CAD; ESA Biosciences,Chelmsford, Mass.) with a nebulizer temperature at 30° C.

Plasma biomarker analysis. Total cholesterol, free cholesterol, andtriglycerides in plasma were determined by enzymatic colorimetric assaysusing a Roche Diagnostics/Hitachi 914 Clinical Analyzer with assay kitsfrom Roche Diagnostics (Indianapolis, Ind.) and Diagnostic Chemicals,Ltd. (Oxford, Conn.). The concentrations of plasma, LDL-cholesterol andHDL-cholesterol were determined using L-type LDL-cholesterol (RocheDiagnostics) and L-type HDL-cholesterol [Wako Chemicals (Richmond, Va.)]assay kits. The VLDL-cholesterol levels were calculated by subtractingHDL-cholesterol and LDL-cholesterol from total cholesterol levels.Plasma concentrations of adiponectin, leptin, and insulin of 12 h-fastedmice were determined using mouse adiponectin (B-Bridge International,Sunnyvale, Calif.), leptin (Assay Designs, Ann Arbor, Mich.), andinsulin (Mercodia Inc., Winston Salem, N.C.) immunoassay kits, asdescribed15. Fasting glucose levels were measured by collecting bloodfrom each mouse by the tail-prick approach. A drop of blood collected bya sterile needle was analyzed using a OneTouch®Ultra® meter withFastDraw™ test strips (Johnson & Johnson, Milpitas, Calif.).

PCR Amplifcation. After genomic DNA extraction and quantification,samples were prepared for amplification and sequencing at the JCVI JointTechnology Center (JTC). Genomic DNA sample concentrations werenormalized to ˜2-6 ng/μl. The V3-V5 region of the 16S rRNA gene wasamplified using forward primer 341F (5′-CCTACGGGAGGCAGCAG-3′ (SEQ ID NO:12)) and reverse primer 926R (5′-CCGTCAATTCMTTTRAGT-3′ (SEQ ID NO: 13)).A barcoded primer design was completed using a set of algorithmsdeveloped at the JCVI. The ‘A’ and ‘B’ adapters for 454 libraryconstruction were included as a part of the PCR primers. To the 926Rprimer, 10 nt barcodes were included as part of the primer design(5′-A-adapter-N(10)+165 primer-3′). This design allowed for theinclusion of a unique barcode to each sample at the time of PCR so thatthe tagged samples could be multiplexed for sequencing. Every effort wasmade to prevent contamination of PCR reactions with exogenous DNAincluding a set of reactions in a laminar flow hood. PCR reactions werecompleted as follows (per reaction): 2 μL of gDNA, 1× finalconcentration of Accuprime PCR Buffer II (Invitrogen, Carlsbad, Calif.,USA), 200 nM forward and reverse primers, 0.75 units of Accuprime TaqDNA Polymerase High Fidelity (Invitrogen, Carlsbad, Calif., USA), andnuclease-free water to bring the final volume to 20 μL. PCR cyclingconditions were: initial denaturation of 2 minutes at 95° C. followed by30 cycles of 20 seconds at 95° C., 30 seconds at 50° C., and 5 minutesat 72° C. A negative control (water blank) reaction also was includedand examined after 35 cycles. PCR reactions were visualized on 1%agarose gels and quantified using a Tecan SpectraFluor Plus (Tecan GroupLtd., Mannedorf, Switzerland). Each reaction was cleaned individuallyusing the Agencourt AMPure system (Beckman Coulter Genomics, DanversMass., USA) prior to normalization and pooling of samples forsequencing.

Sequencing. The pooled samples were further cleaned using the AgencourtAMPure system (Beckman Coulter Genomics, Danvers Mass., USA) prior toemulsification (em)PCR. Steps for emPCR, enrichment and 454 sequencingwere performed by following the vendor's standard operating procedureswith some modifications. Specifically, qPCR was used to accuratelyestimate the number of molecules needed for emPCR. We also utilizedautomation (BioMek FX) to “break” the emulsions after emPCR, and we usedbutanol to enable easier sample handling during the breaking process.

Bioinformatic pipeline 2. The Qiime pipeline (Caporaso et al., NatMethods 7, 335-336, 2010) was used to further process the sequences. Thesequences were first grouped into operational taxonomic units (OTUs)with a sequence similarity threshold of 97%, then taxonomic assignmentwas generated using the RDP database and Qiime algorithm. This data wasused to produce the operational taxonomic unit (OTU) absolute abundancetable and weighted UniFrac beta-diversity matrix (Lozupone et al.,UniFrac: an effective distance metric for microbial communitycomparison. ISME J 2010; Lozupone et al., Appl Environ Microbiol 71,8228-8235, 2005). Principle component analysis (PCA) plots were producedbased on unweighted UniFrac distances. The rarefactions for richness andShannon diversity indices were calculated in R statistical programmingenvironment (R: A Language and Environment for Statistical Computing, inR Foundation for Statistical Computing, Vol. 1, 2009; Gentleman et al.,Genome Biol 5, R80, 2004) using Community Ecology Package vegan.Comparison of unweighted and weighted UniFrac distances was performedusing two-sided t-test. The OTU absolute abundances were converted torelative abundances by normalizing to total sequence count per sampleanalyzed. The resulting relative abundance matrix was used to produceheatmaps for major (relative abundance>1%) taxa.

Comparison of bioinformatic pipeline 2 to bioinformatic pipeline 1 usedin Example 1. The taxonomic data in Example 1 was derived from abioinformatic pipeline that matched the 16S sequence reads to sequencesin the Ribosomal Database Project (RDP) (Wang et al., Applied andEnvironmental Microbiology 73, 5261-5267, 2007) using the RDP classifierwith an 80% confidence. In bioinformatics pipeline 2 analysis (FIGS.7-12), the same sequence data was first grouped into OTU's at a 97%sequence similarity, then assigned a taxonomic name using the Qiimepipeline and RDP database. The taxonomic data varies between the twodata sets generated by the automated RDP classifier (Example 1, FIGS.4-6) and generated by the automated Qiime taxonomic assignment tool,because they represent two independent algorithms that aim to assignmicrobial taxonomic names to sequence data. Diet-mediated alterationsvaried by pipeline at the genus level, but were conserved at the familyand higher levels of classification. Significance testing was done bythe Mann Whitney U test, however the second data set was corrected forfalse discovery rate. This more stringent analysis explains why fewertaxa are revealed as significant changes.

Statistical analysis. All biochemical data are expressed as Scatterplots with means. Differences between dietary groups were evaluatedusing Mann-Whitney U test. Significance was corrected for falsediscovery (Benjamini et al., Journal of the Royal Statistical Society.Series B (Methodological), 289-300, 1995).

Bioinformatic analysis. The Qiime pipeline29 was used to further processthe sequences and produce the operational taxonomic unit (OTU) absoluteabundance table and weighted UniFrac beta-diversity matrix 30,31.Principle component analysis (PCA) plots were produced based on weightedUniFrac distances. The rarefactions for richness and Shannon diversityindices were calculated in R statistical programming environment 32,33using Community Ecology Package vegan. Comparison of weighted UniFracdistances was performed using two-sided t-test. The OTU absoluteabundances were converted to relative abundances by normalizing to totalsequence count per sample analyzed. The resulting relative abundancematrix was used to produce heatmaps for major (relative abundance >1%)taxa.

Results

All C57BL/6J mice were fed a high fat diet (HFD, 60% kcal from fat) for2 weeks prior to the study and then randomized to continue the HFD, toreceive the HFD supplemented with HPMC (HPMC diet), or to receive a lowfat diet (LFD, 10% kcal from fat). Over the course of the 4-week study,the mice on the HFD continued to gain weight, while HPMC supplementationreduced weight gain (FIG. 1A), despite isocaloric food intake (FIG. 1B).Mice receiving the LFD had significantly lower energy intake and lostweight (FIGS. 1A-1B). Weight change and energy intake were correlated inboth the individual HFD and LFD mice (R=0.84 and 0.65, p=0.003 and0.042, respectively); when combined, the correlation strengthened(R=0.98) (FIG. 1C). Based on this best-fit line, the actual weight ofthe HPMC mice was 4.7 g ±2.2 lower than predicted (FIG. 1D), with nosignificant correlation with energy intake. Thus, the HPMC dietdisrupted the energy intake/weight gain relationships observed for thetwo other dietary groups.

After 4 weeks, mice on the LFD and HPMC diets had significantly reducedtotal cholesterol, HDL, LDL, and VLDL, compared to the HFD mice (FIGS.2A-2D). The LFD mice also had decreased fasting blood glucose, andfree-fatty acids (FIGS. 2E, 2G). Leptin levels were decreased in boththe LFD and HPMC mice, consistent with the lowered weights (FIG. 21), aswere liver triglycerides (FIG. 2L). Insulin was decreased in HPMC mice(FIG. 2H). Compared to both the LFD and HFD mice, HPMC supplementationincreased fecal fat excretion, including saturated, unsaturated, andtrans-unsaturated fats, and bile acids, but decreased sterol excretion(FIGS. 2M-2Q). HPMC increased fecal excretion of mono- but not di- ortri-acylglycerides (FIGS. 2R-2T). Thus, in the setting of a high-fat,high-calorie diet, HPMC improved metabolic biomarkers to an extentsimilar to that of the LFD, but with increased fecal loss of specificmetabolites.

Microbial population sizes. To assess quantitative changes in microbialpopulations, total bacteria were enumerated and the two predominantphyla: Bacteroidetes and Firmicutes, using quantitative PCR of 16S rRNAgenes. At baseline, as expected, there were no significant differencesin total fecal bacteria or B/F ratio between the mice about to berandomized into the three treatment groups (FIG. 7). Total bacterialevels were significantly decreased in fecal, cecal, and ileal samplesfrom HPMC mice compared to the two other groups. TheBacteroidetes/Firmicutes (B/F) ratio in fecal samples was unchanged asexpected in the HFD mice, but increased in LFD and HPMC mice and incecal samples from HPMC mice. In contrast, in ileal samples, the B/Fratios were decreased in LFD and HPMC mice (FIG. 7C). These dataindicate that the dietary changes, especially adding HPMC, affected thesize and composition of the intestinal microbiota.

Microbial community composition. To assess changes in murine intestinalmicrobial community structure induced by the dietary changes, measuresof richness and diversity were calculated for microbial 16S rDNA V3-V5sequences in fecal and cecal samples. At the class level, richness inthe fecal and cecal communities from the three groups were similar (FIG.14A), but at the OTU level, the LFD and HPMC mice had decreased fecalcommunity richness compared to HFD, and the HPMC mice had reducedrichness in cecal samples as well (FIG. 15A). Thus, although populationstructure was conserved at the higher (class) taxonomic level, thedietary changes diminished diversity at the more specific (OTU)taxonomic level. Evenness at the class level as measured by Shannonscore, increased in the LFD and HPMC mouse fecal samples, and in cecalsamples for HPMC mice (FIG. 14B), but decreased in the same groups atthe OTU level (FIG. 15B). These findings suggest that the dietaryinterventions favored a relatively small number of specialist organismswithin larger taxonomic units that became more balanced.

The phylogenetic differences between the three treatment groups alsowere assessed by principal component analysis (PCA) based on unweightedUnifrac values (FIG. 16). At week 0 (baseline), all samples clustertogether (FIG. 16A), while at week 2 (FIG. 16B) and week 4 (FIG. 16C),the HFD samples were little different from baseline, whereas the LFD andHPMC communities had shifted in separate directions forming distinctclusters (FIGS. 16D, 16E). There was no significant change in pairwiseUniFrac distances over time for any of the HFD fecal samples. For theLFD samples, pairwise distances increased from weeks 0 to 2, but notfrom 2 to 4. For the HPMC fecal samples, pairwise distances increasedfrom weeks 0 to 2, and from 2 to 4. Analysis of the weighted UniFracdistances showed similar trends (FIG. 17). The PCA of the cecalspecimens obtained at sacrifice is similar to the week 4 fecalspecimens, but the LFD and HFD samples are well-mixed, with HPMC forminga distinct cluster (FIG. 16F). The ileal specimens at sacrifice show nodistinctive clustering by treatment group (FIG. 16G). In total, the dataindicate stability of the microbial community structure in the micecontinued on HFD, as expected, whereas there was an early and persistingshift for the mice given the LFD, and progressive changes for themicrobiota of the mice given HPMC.

Population changes induced by dietary intervention. Hierarchicalclustering by heat map analysis (FIG. 18) for the most abundant taxons(>1%) at the family level in cecal specimens showed clear separation(p<0.001) of the HPMC samples from the HFD and LFD, which were notdistinguishable (FIG. 18A). The fecal samples at week 0 (baseline) werewell-mixed between the three groups, as expected, (FIG. 18B), butcomposition gradually (FIGS. 18C, 18D) moved toward three clusters, witheach treatment group individuating.

To assess specific changes in intestinal microbiota, the rawpyrosequencing counts were converted to relative abundance, and falsediscovery rate (FDR)-corrected pair-wise comparisons for eachpredominant (>1%) taxon from the phylum through genus level were madefor HFD vs LFD (to assess for the effects of fat% dietary change) andHFD vs. HPMC (to assess for the effects of fiber addition) (Table 2). Nosignificant differences were seen in week 0 fecal samples, as expected,since at baseline all mice were receiving the same (HFD) diet. Over thecourse of the experiment, there were no significant differences at thephylum level. All differences at lower taxonomic levels were within thephylum Firmicutes, involving members of class Bacilli, Clostridia, andErysipelotrichi (and order Lactobacillates, Clostridiates, andErysipelotrichales).

At the family level, there were seven predominant taxa (FIG. 10.Lachnospiraceae populations in LFD and HPMC mice became decreased (FIG.10A) and Ruminococcaceae levels became reduced in HPMC mice (FIG. 10B),while Erysipelotrichaceae levels increased in both LFD and HPMC treatedmice (FIG. 10C), and Peptostreptococcaceae levels were consistentlyincreased in HPMC mice (FIG. 10D). Differences in cecal and ilealpopulations were limited to HPMC and LFD mice, respectively. At thegenus level, there also were significant changes in community structure(FIG. 11). The HPMC treatment led to significant decreases inJohnsonella and Lactobacillus, and significant increases inErysipelotrichaceae Insertae Sedis and Peptostreptococcus InsertaeSedis. Thus, the dietary changes selected for different compositionswithin Firmicutes, with reproducible compositional effects at the familylevel.

TABLE 2 Composition of the three experimental diets Low-Fat High-FatHigh-Fat/10% HPMC Component gm % kcal % gm % kcal % gm % kcal % Protein19.2 20 26.2 20 23.6 20 Carbohydrate 67.3 70 26.3 20 23.6 20 Fat 4.3 1034.9 60 31.3 60 Total 100 100 100 kcal/gm 3.85 5.24 4.71 Ingredient gmkcal gm kcal gm kcal Casein, 200 800 200 800 200 800 80 Mesh L-Cystine 312 3 12 3 12 Corn starch 315 1260 0 0 0 0 Maltodextrin 35 140 125 500125 500 10 Sucrose 350 1400 68.8 275.2 68.8 275.2 Cellulose, 50 0 50 050 0 BW200 Soybean Oil 25 225 25 225 25 225 Lard 20 180 245 2205 2452205 Mineral Mix 10 0 10 0 10 0 S10026 Dicalcium 13 0 13 0 13 0Phosphate Calcium 5.5 0 5.5 0 5.5 0 Carbonate Potassium 16.5 0 16.5 016.5 0 Citrate, 1 H2O Vitamin 10 40 10 40 10 40 Mix V10001 Choline 2 0 20 2 0 Bitartrate HPMC^(a) 0 0 0 0 88 0 FD&C 0.05 0 0 0 0.025 0 YellowDye #5 FD&C 0 0 0.05 0 0.025 0 Blue Dye #1 ^(a)HPMC, hydroxyl-methylcellulose (K250 M)

Correlation Network Analysis. To understand the dynamic relationshipsbetween intestinal microbiota under differing dietary conditions, acorrelation network was constructed at the order level (FIG. 20). Atbaseline (week 0), Clostridiales significantly negatively correlatedwith Lactobacillales in all three groups of mice and with Bacteroidalesin two of the three groups. The negative correlations between thesethree taxa persisted in the HFD mice over the 4-week study. However,both dietary interventions (LFD and HPMC) eliminated the stable linkbetween Bacteroidales-Clostridiales-Lactobacillales. LFD mice showed astable negative correlation between Clostridiales andErysipelotrichales, whereas no significant correlations emerged in theHPMC mice. Thus, the major bacterial networks established under HFDconditions were not conserved after dietary change.

Conditional Correlation Analysis. The results show that HPMC or LFDtreatments altered both host metabolism and intestinal microbiota. Todetect individual taxa that may be responsible for metabolic effects,conditional correlation analysis was performed, to remove the effect ofthe treatment condition (either presence of fiber or fat percent) fromthe two linked variables: host phenotype and taxa. SignificantFDR-corrected p-values for each pairwise comparison of taxa inparticular samples with each metabolic variable are indicated (Tables3-4) and regression analysis was used to avoid reporting correlationsinfluenced by outlier samples (FIG. 21. Weight change in HFD mice waspositively correlated with cecal Firmicutes and cecalErysipelotrichaceae Insertae Sedis (I. S.) and negatively associatedwith cecal Bacteroidetes (FIGS. 21A-21C). For the HPMC mice, weightchanges and fecal saturated fat were positively associated with cecalErysipelotrichaceae I.S. and cecal Erisipelotrichales, respectively(FIGS. 21C, 21F). Four-week fecal abundances of Lachnospiraceaenegatively correlated with energy intake in the HFD and HPMC mice (FIG.21D and FIG. 23) and cecal Porphyromonadaceae correlated positively withliver free cholesterol in HFD and HPMC mice (FIG. 21E). In total, therewere no significant correlations between taxa and host metabolicphenotype for the LFD group, however, the HPMC treatment-induced hostphenotypes correlated with altered representation of particular taxa.

Table 3 shows p-values calculated for false-discovery corrections fromMann Whitney U-pair comparisons of relative abundance. Sample typesincluded week 0 fecal (baseline, 0F), week 2 fecal (2F), week 4 fecal(4F), wee 4 cecal (4C), week 4 ileal (41I).

p-values <0.05 are shaded in grey.

Analysis of Erysipelotrichaceae Incertae Sedis

Sequences that are categorized at the genus level withinErysipelotrichaceae incertae sedis (EIS) account for 4.5% of allmicrobial 16S rRNA sequences in the present study. Incertae sedis inLatin means uncertain seat and notates a placeholder for a potential newgenus in which defined species and sequences of uncultivated organismsare placed. A list of cultured and named species in unclassifiedErysipelotrichaceae, according to NCBI taxonomy is shown below:.

Clostridium cocleatum Clostridium innocuum Clostridium ramosumClostridium spiraforme Eubacterium bioforme Eubacterium cylindroidesEubacterium dolichum Eubacterium tortuosum Lactobacillus catenaformisLactobacillus vitulinus Streptococcus pleomorphus

Erysipelotricaceae Incertae Sedis (EIS) is a taxon of interest becauseit is increased significantly in the HPMC-treated mice (FIG. 12). Toinvestigate the potential identity of members of EIS, the 109 uniquesequences assigned to EIS at the OTU level were aligned using Greengenesto find the nearest neighbor. Minimum length was set at 100 nucleotidesand minimum percent identity was set at 75%. There were 24 uniqueorganisms identified as closest matches.

59 of the 109 EIS OTUs had a closest match to Clostridium cocleatum str.DSM 1551, accounting for 62.1% of the EIS sequences and 2.8% of thetotal taxonomic sequences from the 454-pyrosequencing run. C. cocleatumhad an average sequence identity of 95.5% (±0.28% S.E., min 89.4%, max98.6%) over an average sequence length of 367 nucleotides (±11.3 S.E,min 108, max 489). One OTU comprised 20.0% of the EIS sequences, and0.9% of the total taxonomic sequences, and had a closest match touranium-contaminated aquifer clone 1013-28-CG45 (UCAC), classified inRDP as Firmicutes; Clostridia; Clostridiales; Peptococcaceae;Peptococcaceae 1; Desulfosporosinus, with a sequence identity of 93.7%over a length of 270 nucleotides. All other closest matches accountedfor less than 0.2% of the total taxonomic sequences, and less than 4% ofthe EIS sequences (Table 5). Due to the high relative abundance (FIG.24) and high percent identity of OTUs to Clostridium cocleatum, thisorganism is a promising candidate.

TABLE 6 Identity of closest matches of OTUs classified asErysipelotrichaceae incertae sedis (EIS) determined by sequence BLASTusing GreenGenes. The first column indicates relative abundance in all147 samples from the Diet/Fiber study and the second column is % ofsequences classified as EIS. % of Average BLAST % of totalErysipelotrichaceae percent identity to sequences incertae sedistemplate #OTUs Closest Match 2.77 62.14 95.5 59 Clostridium cocleatumstr. DSM 1551 0.89 20.00 93.7 1 uranium-contaminated aquifer clone1013-28- CG45 0.14 3.16 81.6 1 marine bone clone boneC3C9 0.12 2.68 86.35 human fecal clone RL305aal86g01 0.12 2.65 88.9 7 culturable kangarooMacropus giganteus Eastern grey Kangaroo forestomach contents 0.10 2.3584.6 2 Caminicella sporogenes str. AM1114 0.10 2.16 90.5 5 Coprobacilluscateniformis str. JCM 10605 0.08 1.73 83.8 1 mouse cecum cloneSWPT19_aaa04g11 0.03 0.74 82.7 2 human fecal clone RL197_aah85c11 0.020.50 88.3 2 swine intestine clone p-2772-24E5 0.02 0.38 80.4 1Eubacterium cylindroides str. JCM 7786 0.01 0.26 87.1 2 Spiroplasmainsolitum str. ATCC 33502; M55 0.01 0.25 89.2 3 human fecal cloneRL117_aae92d08 0.01 0.21 94.0 7 Eubacterium sp. Pei061 0.01 0.18 82.0 1sediment-free PCB-dechlorinating enrichment culture clone JN18_A14_H0.01 0.16 88.2 2 mesophilic anaerobic digester clone G35_D8_H_B_E070.004 0.10 84.7 1 human fecal clone RL179aan75d04 0.004 0.09 86.0 1Catenibacterium mitsuokai str. JCM 10609 0.003 0.08 87.0 1 human fecalclone RL200_aai60f02 0.003 0.07 84.2 1 Electricigen Enrichment MFCfull-scale anaerobic bioreactor sludge treating brewery waste clone31a07 0.003 0.06 86.2 1 Inhibition nitrate reduction chromium (VI)microcosms heavy metal contaminated anaerobic soil microcosm isolateGNCr-2GNCr-2 str. GNCr-2 0.002 0.04 87.2 1 Enterococcus mundtii str.NFRI 7393 0.001 0.02 91.7 1 human fecal clone RL176_aan58b04 0.004 0.0288.8 1 mouse cecum clone M2_d06 4.46 100.00 109 ← Total

Conclusions

The present study demonstrates that reducing dietary fat, or addingdietary fiber improves markers of metabolic health in mice receiving ahigh-calorie/high-fat diet. In particular, this study shows that addingHPMC disrupts a strong relationship between energy intake and weightchange. One explanation for the observed deviation may be the increasedexcretion of bile salts and fats in the feces induced by HPMC,representing a form of energy wasting. As shown herein, changing fromHFD to a LFD, or adding 10% HPMC to HFD resulted in marked shifts in theintestinal microbiota over a 4-week period, providing evidence that HPMCalters the intestinal microbiota. The results are highly consistentwithin the experimental groups and show progressive changes in themicrobiota sampled in the feces, with consistent shifts in the cecal andileal samples collected at sacrifice.

HPMC and LFD both improve metabolic health and shape the microbiome,however linkages between specific microbial taxa and host metabolicphenotypes could only be identified in HPMC treated mice and not in LFDmice. This dichotomy suggests that the improvement in health by HPMC ismediated by the intestinal microbiota, while health benefits achieved byLFD are independent of gut microbes. Since HPMC is assumed to be inert,and not acted on by intestinal microbiota, how could this be achieved?

One possible mechanism of action related to HPMC effects on the gutmicrobiome could be the reduction in microbial load. Although changingfrom HFD to LFD did not affect total bacterial densities, adding HPMCclearly had a lowering effect and modulated overall composition asobserved in essentially every analysis. Although ecological richnessshowed no changes at higher taxonomic orders, richness of theHPMC-impacted intestinal community and evenness at the OTU (species)level declined. Such dynamics are consistent with HPMC selection for asmall group of specialist organisms that are over-represented at theexpense of the usual community members.

The extent of the dynamics swings, as detailed in studies of communitystructure that are based on phylogenetic relationships among the taxaand shown by PCA presentations of the Unifrac analyses (FIGS. 16-17) andby heat map (FIG. 18) indicate HPMC-induced progressive shifts incommunity structure in fecal, as well as in cecal, populations. Changingfrom HFD to LFD also produces shifts in community composition, but inways that differ from the HPMC-induced shifts.

All of the major HPMC-induced shifts involve families within Firmicutes,with major decreases in Lachnospiraceae and Ruminococcaceae, andincreases in Erysipelotrichaceae and Peptostreptococcaceae (FIG. 10).That Firmicutes but not Bacteroidetes are affected indicates differencesin their functional or anatomic niches worth future exploration; in thissense, HPMC treatment is a probe of intestinal microbiome populationstructure. Based on the multiple specimens obtained from mice receivingthe HFD, there are conserved numerical relationships among order-leveltaxa (FIG. 20) that are disrupted after HPMC is added. Informaticanalysis indicated conserved patterns of co-variance among major taxa inthe baseline and continuing HFD mice. Importantly, change to HMPC andLFD diet ablated the prior co-variances, and a new relationship wasestablished in LFD mice.

Importantly from this work, we now identify specific taxa that areassociated with weight gain (e.g. Erysipelotrichaceae Incertae Sedis(EIS)), and with other metabolic phenotypes (FIG. 21 and Tables 4-5).

Without wishing to be bound by a specific theory, HPMC could alter themicrobial ecosystem in several ways: (i) HPMC may bind to mucusglycoproteins 22-24 and swell in the large intestine, potentiallydisplacing mucosa-associated microbiota. (ii) HPMC may sequesterglycoside hydrolases or other catalytic enzymes, by mimicking thecarbohydrate structures to which they bind, decreasing microbiomecapacity for energy harvest from complex carbohydrates (for example,HPMC, resembling the structural subunit of amylase, has been used totrap and recover (β-amylase from Clostridium thermosulfurongenes 25),and (iii) such sequestration may affect the balance between bile saltexcretion and reabsorption affecting cholesterol and fat excretion. (iv)HPMC may be partially fermented in the intestine, stimulating the growthof organisms that digest cellulose or secondary metabolites from HPMC.

Example 3 Testing of Probiotic Compositions of the Invention in MouseModels Methods and Abbreviations

Abbreviations. All abbreviations also apply to FIGS. 22-24. CSHEPCc=atleast one of Coprobacillus, Sporacetigenium, Holdemania. JO=at least oneof Johnsonella, Oscillibacter. Prebiotic=at least one of trehalose,cellobose, maltose, mannose, sucrose, lactose, salicin, mellibiose,raffinose, galactose, and fructose. C57B6 mice=C57/B16J mice(approximately 2 months old in the beginning of the experiment).

3.1 Determination of the Effect of CSHEPCc and/or Prebiotic Treatment onWeight Gain in a DIO Mouse Model

As schematically shown in FIG. 22, four groups of C57/B16J mice (atleast 10 mice in each group) are fed 60% fat (DIO) diet from two monthsto four months of age. At four months of age, group 1 is kept untreated(control), group 2 gets CSHEPCc delivered by gastric gavage(Coprobacillus, Holdemania, and Peptostreptococcacea Incertae Sedis aredelivered as live cells, Sporacetigenium, Erysipelotrichaceae IncertaeSedis, and Clostridium cocleatum are delivered as spores; at a dose of10⁹ cells each week), group 3 gets prebiotic only (at a dose of 10 g/Lin the drinking water every day), group 4 gets CSHEPCc+Prebiotic.

Measurements:

-   Genotype: qPCRs for Eubacteria, Firmicutes, Bacteroidetes, BCoAT,    and individual qPCRs for C, S, H, E, P, Cc, J, O-   Phenotype: weight every week, fasting glucose, oral glucose    tolerance test, insulin tolerance test, total cholesterol, LDL, HDL,    triglycerides (TGA), dual emission X-ray absorptiometry (DEXA) every    2 weeks to examine total weight, lean composition, bone mineral    density, and fat composition (and percent fat); fecal short-chain    fatty acids (SCFA), including butyrate and acetate); fasting serum    leptin, ghrelin, insulin, GIP (glucose-dependent insulinotropic    peptide), CRP (C-Reactive Protein).

3.2 Determination of the Effect of CSHEPCc and/or Prebiotic Treatment onPreventing Obesity in a DIO Mouse Model

As shown in FIG. 23, in this experiment, eighty (80) C57/B16J mice arefed 10% fat diet until they are two months old. At four weeks of age(while on 10% diet), mice are divided into four treatment groups: group1 is kept untreated (control), group 2 gets CSH delivered by gastricgavage (Coprobacillus, Holdemania, and Peptostreptococcaceae aredelivered as live cells, Sporacetigenium, Erysipelotrichaceae IncertaeSedis, and Clostridium cocleatum are delivered as spores; at a dose of10⁹ cells of each every week), group 3 gets prebiotic only (at a dose of10 g/L in the drinking water every day), group 4 gets CSHEPCc+Prebiotic.At two months of life, half of the mice (10 mice) in each treatmentgroup are switched to 60% DIO diet, and half of the mice (10 mice) arekept on 10% diet. The same genotypes and phenotype parameters aremeasured as specified in section 3.1, above.

3.3 Determination of the Effect of JO and/or CSHEPCc Treatment on WeightGain and Prevention of Obesity in a Germ-Free Mouse Model

As shown in FIG. 24, in this experiment, germ-free (GF) C57/Black 6 miceare generated and treated with: (1) CSHEPCc as monocultures (one, two,three, or four organisms at a time) or with JO as monocultures (J or O);(2) altered Schaedler flora (ASF) which consists of eight common mouseintestinal commensal bacteria which were developed to colonize germ-freemice and to establish a uniform baseline of conventionally colonizedmice (Dewhirst et at., Appl. Environ. Microbiol., 1999, 65:3287-3292)followed by introduction of CSHEPCc as monocultures or JO (J or O), ornothing (control); (3) feces from normal mice treated with CSHEPCc orcontrol untreated normal mice. From two months of age to four months ofage, mice are fed a 10% fat diet. At four months of age, half of themice (10 mice) in each treatment group are switched to the 60% DIO diet,and half of the mice (10 mice) are kept on the 10% diet. The samegenotypes and phenotype parameters are measured as specified in section3.2, above.

REFERENCES

-   1. Reppas, C., Swidan, S. Z., Tobey, S. W., Turowski, M. &    Dressman, J. B.

Hydroxypropylmethylcellulose significantly lowers blood cholesterol inmildly hypercholesterolemic human subjects. Europ J Clin Nutri 63, 71-77(2009). (Need better ref)

-   2. Carr, T., Gallaher, D., Yang, C. & Hassel, C. Increased    intestinal contents viscosity reduces cholesterol absorption    efficiency in hamsters fed hydroxypropyl methylcellulose. J of Nutri    126, 1463 (1996). (Need better ref)-   3. Gallaher, D., Hassel, C. & Lee, K. Relationships between    viscosity of hydroxypropyl methylcellulose and plasma cholesterol in    hamsters. J of Nutri 123, 1732 (1993).-   4. Maki, K. C., et al. High-viscosity hydroxypropylmethylcellulose    blunts postprandial glucose and insulin responses. Diabetes Care 30,    1039-1043 (2007).-   5. Maki, K. C., et al. Dose-response characteristics of    high-viscosity hydroxypropylmethylcellulose in subjects at risk for    the development of type 2 diabetes mellitus. Diabetes Technol Ther    11, 119-125 (2009).-   6. Maki, K. C., et al. Hydroxypropylmethylcellulose and    methylcellulose consumption reduce postprandial insulinemia in    overweight and obese men and women. J Nutr 138, 292-296 (2008).-   7. Qin, J., et al. A human gut microbial gene catalogue established    by metagenomic sequencing. Nature 464, 59-65 (2010).-   8. Wong, J. M. W., de Souza, R., Kendall, C. W. C., Emam, A. &    Jenkins, D. J. A. Colonic health: fermentation and short chain fatty    acids. Journal of Clinical Gastroenterology 40, 235-243 (2006).-   9. Louis, P., et al. Restricted distribution of the butyrate kinase    pathway among butyrate-producing bacteria from the human colon.    Journal of Bacteriology 186, 2099-2106 (2004).-   10. Savage, D. C. Microbial ecology of the gastrointestinal tract.    Annu Rev Microbiol 31, 107-133 (1977).-   11. Turnbaugh, P. J., et al. An obesity-associated gut microbiome    with increased capacity for energy harvest. Nature 444, 1027-1131    (2006).-   12. Savage, D. C. Gastrointestinal microflora in mammalian    nutrition. Annu Rev Nutr 6, 155-178 (1986).-   13. Siepmann, J. & Peppas, N. A. Modeling of drug release from    delivery systems based on hydroxypropyl methylcellulose (HPMC). Adv    Drug Deliv Rev 48, 139-157 (2001).-   14. Gallaher, D. D., Hassel, C. A., Lee, K. J. & Gallaher, C. M.    Viscosity and fermentability as attributes of dietary fiber    responsible for the hypocholesterolemic effect in hamsters. J Nutr    123, 244-252 (1993).-   15. Hung, S.-C., et al. Dietary fiber improves lipid homeostasis and    modulates adipocytokines in hamsters. J Diabetes 1, 194-206 (2009).-   16. Topping, D. Hydroxypropylmethylcellulose, viscosity, and plasma    cholesterol control. Nutr Rev 52, 176-178 (1994).-   17. Cook, S. I. & Sellin, J. H. Review article: short chain fatty    acids in health and disease. Aliment Pharmacol Ther 12, 499-507    (1998).-   18. Braun, W. H., Ramsey, J. C. & Gehring, P. J. The lack of    significant absorption of methylcellulose, viscosity 3300 CP, from    the gastrointestinal tract following single and multiple oral doses    to the rat. Food Cosmet Toxicol 12, 373-376 (1974).-   19. Machle, W., Heyroth, F. & Witherup, S. The Fate of    Methylcellulose in the Human Digestive Tract. 1-9 (1944).-   20. Yokoyama, W., Knuckles, B., Davis, P. & Daggy, B. Stability of    ingested methylcellulose in the rat determined by polymer molar mass    measurements by light scattering. Journal of agricultural and food    chemistry 50, 7726-7730 (2002).-   21. Ferguson, M. & Jones, G. Production of short chain fatty acids    following in vitro fermentation of saccharides, saccharide esters,    fructo oligosaccharides, starches, modified starches and non starch    polysaccharides. Journal of the Science of Food and Agriculture 80,    166-170 (2000).-   22. Cai, X., Yang, L., Zhang, L.-M. & Wu, Q. Synthesis and anaerobic    biodegradation of indomethacin-conjugated cellulose ethers used for    colon-specific drug delivery. Bioresource Technology 100, 4164-4170    (2009).-   23. Haupt, S. & Rubinstein, A. The colon as a possible target for    orally administered peptide and protein drugs. Critical reviews in    therapeutic drug carrier systems 19, 499 (2002).-   24. Hamman, J. H., Enslin, G. M. & Kotze, A. F. Oral delivery of    peptide drugs: barriers and developments. BioDrugs 19, 165-177    (2005).-   25. Miranda, E. & Berglund, K. Recovery of Clostridium    thermosulfurogenes produced.

beta.-amylase by hydroxypropyl methylcellulose partition. BiotechnologyProgress 6, 214-219 (1990).

-   26. Clark, J., et al. Guide for the care and use of laboratory    animals. Institute of Laboratory Animal Resources, National Research    Council, Washington, DC (1996).-   27. AOCS. Preparation of methyl esters of long-chain fatty acids    from sampling analysis of commercial fats and oils. Official method,    AOCS, Champaign, Ill. Ce 2-66 (1997).-   28. Hong, Y. J., Turowski, M., Lin, J. T. & Yokoyama, W. H.    Simultaneous characterization of bile acid, sterols, and    determination of acylglycerides in feces from soluble cellulose-fed    hamsters using HPLC with evaporative light-scattering detection and    APCIñMS. Journal of agricultural and food chemistry 55, 9750-9757    (2007).-   29. Caporaso, J. G., et al. QIIME allows analysis of high-throughput    community sequencing data. Nat Methods 7, 335-336 (2010). 30.    Lozupone, C., Lladser, M. E., Knights, D., Stombaugh, J. &    Knight, R. UniFrac: an effective distance metric for microbial    community comparison. ISME J(2010).-   31. Lozupone, C. & Knight, R. UniFrac: a new phylogenetic method for    comparing microbial communities. Appl Environ Microbiol 71,    8228-8235 (2005).-   32. R: A Language and Environment for Statistical Computing. in R    Foundation for Statistical Computing, Vol. 1 (R Foundation for    Statistical Computing, 2009).-   33. Gentleman, R. C., et al. Bioconductor: open software development    for computational biology and bioinformatics. Genome Biol 5, R80    (2004).-   34. Arumugam M, Raes J, Pelletier E, Le Pastier D, et al.    Enterotypes of the human gut microbiome. Nature 2011;473:174-80.-   35. Muegge B D, Kuczynski J, Knights D, Clemente J C, González A,    Fontana L, Henrissat B, Knight R, Gordon J I. Diet drives    convergence in gut microbiome functions across mammalian phylogeny    and within humans. Science 2011; 332:970-4.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and theaccompanying figures. Such modifications are intended to fall within thescope of the appended claims. It is further to be understood that allvalues are approximate, and are provided for description.

Patents, patent applications, publications, product descriptions, andprotocols are cited throughout this application, the disclosures ofwhich are incorporated herein by reference in their entireties for allpurposes.

1.-19. (canceled)
 20. A method for promoting weight loss or preventingweight gain in a mammal in need thereof comprising administering to themammal a therapeutically effective amount of a probiotic composition,wherein said probiotic composition stimulates growth or metabolicactivity of at least one strain from the taxon selected from the groupconsisting of Coprobacillus, Sporacetigenium, Holdemania, Dorea,Blautia, Enterococcus, Erysipelotrichaceae, Clostridium cocleatum, andPeptosteptococcaceae IS (PIS) in the intestinal microbiota of themammal.
 21. A method for preventing or treating a disease in a mammal inneed thereof, wherein the disease is selected from the group consistingof obesity, metabolic syndrome, diabetes mellitus, insulin-deficiencyrelated disorders, insulin-resistance related disorders, glucoseintolerance, non-alcoholic fatty liver, abnormal lipid metabolism, andatherosclerosis, said method comprising administering to the mammal atherapeutically effective amount of a probiotic composition, whereinsaid probiotic composition stimulates growth or metabolic activity of atleast one strain from the taxon selected from the group consisting ofCoprobacillus, Sporacetigenium, Holdemania, Dorea, Blautia,Enterococcus, Erysipelotrichaceae, Clostridium cocleatum, andPeptosteptococcaceae IS (PIS) in the intestinal microbiota of themammal. 22-27. (canceled)
 28. A method for promoting weight loss orpreventing weight gain in a mammal in need thereof comprisingadministering to the mammal (i) a therapeutically effective amount of anarrow spectrum antibiotic or (ii) a therapeutically effective amount ofa probiotic composition, wherein said narrow spectrum antibiotic orprobiotic composition inhibits growth or metabolic activity of at leastone strain from the taxon selected from the group consisting ofJohnsonella, Oscillibacter, Lachnospiraceae, Ruminococcaceae, andClostridiales in the intestinal microbiota of the mammal.
 29. A methodfor preventing or treating a disease in a mammal in need thereof,wherein the disease is selected from the group consisting of obesity,metabolic syndrome, diabetes mellitus, insulin-deficiency relateddisorders, insulin-resistance related disorders, glucose intolerance,non-alcoholic fatty liver, abnormal lipid metabolism, andatherosclerosis, said method comprising administering to the mammal (i)a therapeutically effective amount of a narrow spectrum antibiotic or(ii) a therapeutically effective amount of a probiotic composition,wherein said narrow spectrum antibiotic or probiotic compositioninhibits growth or activity of at least one strain from the taxonselected from the group consisting of Johnsonella, Oscillibacter,Lachnospiraceae, Ruminococcaceae, and Clostridiales in the intestinalmicrobiota of the mammal.
 30. (canceled)
 31. The method of claim 28,wherein the probiotic composition comprises at least one strain from thetaxon selected from the group consisting of Coprobacillus,Sporacetigenium, Holdemania, Dorea, Blautia, Enterococcus,Erysipelotrichaceae, Clostridium cocleatum, and Peptosteptococcaceae IS(PIS). 32-119. (canceled)
 120. The method of claim 20, wherein theprobiotic composition comprises at least one strain from the taxonselected from the group consisting of Coprobacillus, Sporacetigenium,Holdemania, Blautia, Enterococcus, Erysipelotrichaceae, Clostridiumcocleatum, and Peptosteptococcaceae IS (PIS).
 121. The method of claim120, wherein said strain is selected from the group consisting of livebacterial strains, spores and conditionally lethal bacterial strains.122. The method of claim 20, wherein the probiotic composition isadministered conjointly with a prebiotic composition which stimulatesgrowth and/or metabolic activity of bacteria contained in the probioticcomposition.
 123. The method of claim 122, wherein the probiotic andprebiotic compositions are administered in one composition, orsimultaneously as two separate compositions, or sequentially.
 124. Themethod of claim 20, wherein the probiotic composition is administeredorally or rectally.
 125. The method of claim 20, wherein the probioticcomposition is administered in a form of a capsule or in a form of asuppository.
 126. The method of claim 20, wherein the mammal is human.127. The method of claim 21, wherein the probiotic composition comprisesat least one strain from the taxon selected from the group consisting ofCoprobacillus, Sporacetigenium, Holdemania, Dorea, Blautia,Enterococcus, Erysipelotrichaceae, Clostridium cocleatum, andPeptosteptococcaceae IS (PIS).
 128. The method of claim 127, whereinsaid strain is selected from the group consisting of live bacterialstrains, spores and conditionally lethal bacterial strains.
 129. Themethod of claim 21, wherein the probiotic composition is administeredconjointly with a prebiotic composition which stimulates growth and/ormetabolic activity of bacteria contained in the probiotic composition.130. The method of claim 129, wherein the probiotic and prebioticcompositions are administered in one composition, or simultaneously astwo separate compositions, or sequentially.
 131. The method of claim 21,wherein the probiotic composition is administered orally or rectally.132. The method of claim 21, wherein the probiotic composition isadministered in a form of a capsule or in a form of a suppository. 133.The method of claim 21, wherein the mammal is human.